DNA encoding mammalian neuropeptide FF (NPFF) receptors and uses thereof

ABSTRACT

This invention provides isolated nucleic acids encoding mammalian NPFF receptors, purified mammalian NPFF receptors, vectors comprising nucleic acid encoding mammalian NPFF receptors, cells comprising such vectors, antibodies directed to mammalian NPFF receptors, nucleic acid probes useful for detecting nucleic acid encoding mammalian NPFF receptors, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding mammalian NPFF receptors, transgenic, nonhuman animals which express DNA encoding normal or mutant mammalian NPFF receptors, methods of isolating mammalian NPFF receptors, methods of treating an abnormality that is linked to the activity of the mammalian NPFF receptors, as well as methods of determining binding of compounds to mammalian NPFF receptors, methods of identifying agonists and antagonists of NPFF receptors, and agonists and antagonists so identified.

BACKGROUND OF THE INVENTION

This application is a continuation of U.S. Ser. No. 09/538,036, filedMar. 29, 2000, now U.S. Pat. No. 6,849,727, issued Feb. 1, 2005, whichis a continuation-in-part of U.S. Ser. No. 09/405,558, filed Sep. 24,1999, now U.S. Pat. No. 6,709,831 B1, issued Mar. 23, 2004, which is acontinuation-in-part of U.S. Ser. No. 09/255,368, filed Feb. 22, 1999,now U.S. Pat. No. 6,262,246 B1, issued Jul. 17, 2001, which is acontinuation-in-part of U.S. Ser. No. 09/161,113, filed Sep. 25, 1998,now abandoned, the contents of all of which are hereby incorporated byreference into the subject application.

Throughout this application, various publications are referenced inparentheses. Full citations for these references may be found at the endof the specification immediately preceding the sequence listings and theclaims. The disclosure of these publications in their entireties arehereby incorporated by reference into this application to describe morefully the art to which this invention pertains.

Neuroregulators comprise a diverse group of natural products thatsubserve or modulate communication in the nervous system. They include,but are not limited to, neuropeptides, amino acids, biogenic amines,lipids and lipid metabolites, and other metabolic byproducts. Many ofthese neuroregulator substances interact with specific cell surfacereceptors which transduce signals from the outside to the inside of thecell. G-protein coupled receptors (GPCRs) represent a major class ofcell surface receptors with which many neurotransmitters interact tomediate their effects. GPCRs are predicted to have sevenmembrane-spanning domains and are coupled to their effectors via

G-proteins linking receptor activation with intracellular biochemicalsequelae such as stimulation of adenylyl cyclase.

Neuropeptide FF (NPFF) is an octapeptide isolated from bovine brain in1985 by Yang and coworkers (1) using antibodies to the molluscanneuropeptide FMRFamide (FMRFa). FMRFamide-like immunoreactivity wasobserved in rat brain, spinal cord, and pituitary, suggesting theexistence of mammalian homologs of the FMRFa family of invertebratepeptides. The isolation of NPFF, named for its N- and C-terminalphenylalanines (also called F8Famide) and a second mammalian peptide,NPAF (also called A18Famide), confirmed the existence of mammalianfamily of peptides sharing C-terminal sequence homology with FMRFa (1).Molecular cloning has revealed that NPFF and NPAF are encoded by thesame gene and cleaved from a common precursor protein (2). Studies ofthe localization, radioligand binding, and function of NPFF-likepeptides (see below) indicate they are neuromodulatory peptides whoseeffects are likely to be mediated by G protein-coupled receptors (forreview, see 3).

NPFF, also called “morphine modulating peptide”, is an endogenousmodulator of opioid systems with effects on morphine analgesia,tolerance, and withdrawal (for review see 3,4). NPFF appears torepresent an endogenous “anti-opioid” system in the CNS acting atspecific, high-affinity receptors distinct from opiate receptors (5,6).Endogenous NPFF has been suggested to play a role in morphine tolerance:agonists of NPFF precipitate “morphine abstinence syndrome” (i.e.symptoms of morphine withdrawal) in morphine-dependent animals (7,8),while antagonists and anti-NPFF IgG restore morphine sensitivity andameliorate symptoms of withdrawal (9-12). NPFF antagonists potentiallycould be useful as therapeutic agents to prevent the development ofmorphine tolerance, and to treat opiate addiction. NPFF has also beensuggested to participate in the regulation of pain threshold, showingboth “anti-opiate” effects and analgesic effects depending on testsystem and route of administration (for review, see 4). As ananti-opiate, NPFF has been shown to inhibit morphine- and stress-inducedanalgesia (1, 13, 14, 15), whereas anti-NPFF IgG (which blocks thebiological activity of NPFF) potentiates these two phenomena (16, 17).An NPFF antagonist may be clinically useful in potentiating theanalgesic effects of morphine, allowing use of lower doses without thedevelopment of tolerance. NPFF agonists may also exhibit analgesicactivity in some model systems (14, 18, 19). The analgesia elicited byNPFF is typically sensitive to naloxone, indicating that it is mediatedby release of endogenous opioid peptides (19, 20). The interaction ofNPFF and opioid systems in regulating pain pathways is thus complex andmay involve multiple mechanisms and sites of action. NPFF has additionalbiological activities in accord with its pattern of expression in thenervous system.

NPFF peptide localization in rat CNS was examined using specificantibodies ((21-23); see also (3)). The highest levels of NPFF are foundin spinal cord and posterior pituitary; pituitary NPFF is believed tooriginate in the hypothalamus. In the brain, immunoreactive cell bodiesare found in two major cell groups: medial hypothalamus (betweendorsomedial and ventromedial) and nucleus of the solitary tract.Immunoreactive fibers are observed in lateral septal nucleus, amygdala,hypothalamus, nucleus of solitary tract, ventral medulla, trigeminalcomplex, and dorsal horn of spinal cord. This localization pattern isconsistent with a role for NPFF in sensory processing and modulation ofopioid systems. In addition, its presence in the hypothalamus and otherlimbic structures could subserve roles in the regulation of appetitiveand affective states. In the periphery, NPFF-like immunoreactivity (aswell as NPFF binding) has been observed in the heart (24). In addition,injection of NPFF raises blood pressure in rats (24, 25). Theseobservations, combined with the colocalization of NPFF withcatecholaminergic neurons in the nucleus of the solitary tract (26),suggest that NPFF is involved in central and peripheral cardiovascularregulation.

The ability of NPFF peptides to modulate the opioid system raised thepossibility that NPFF interacts directly with opiate receptors. However,radioligand binding assays using a tyrosine-substituted NPFF analog[¹²⁵I]Y8Fa demonstrate that NPFF acts through specific high affinitybinding sites distinct from opiate receptors (27-30) that are sensitiveto inhibition by guanine nucleotides (31). The latter observationindicates that NPFF receptors are likely to belong to the superfamily ofG protein-coupled receptors which share common structural motifs.However, no reports of cloning NPFF receptors have appeared as yet.

To address the issue of potential degradation of the peptideradioligand, a more stable NPFF analog (called (1DMe)Y8Fa(18)) has alsobeen radioiodinated and the binding characterized in spinal cordmembranes (32). The binding was saturable and of high affinity;inhibition of binding with unlabeled NPFF analogs yielded Ki values of0.16 nM and 0.29 nM for (1DMe)Y8Fa and NPFF, respectively, with aBmax=15 fmol/mg protein. No inhibition by various opioid compounds(naloxone, morphine, enkephalins, dynorphins, etc.) or other peptides(NPY, SP, CGRP, for examples) was observed at a concentration of 10 μM,confirming the specificity of NPFF receptors. Interestingly, the relatedmolluscan peptide FMRFa inhibited the binding of [¹²⁵I] (1DMe)Y8Fa witha Ki 30 nM. The effectiveness of FMRFamide and the C-terminal fragmentNPFF(6-8) at NPFF receptors suggests an important role for the commonC-terminus. Full activity is retained by NPFF (3-8); it has beensuggested that although the C-terminus is important for receptorrecognition, the N-terminus is necessary for formation of ahigh-affinity conformation (33).

Allard et al. (29) examined the distribution of NPFF binding sites inrat brain and spinal cord using [¹²⁵I]Y8Fa. The highest densities wereobserved in the external layers of dorsal horn of spinal cord, severalbrainstem nuclei, the suprachiasmatic nucleus, restricted nuclei of thethalamus, and the presubiculum of the hippocampus. Lower densities wereseen in central gray, reticular formation, ventral tegmental area,lateral and anterior hypothalamus, medial preoptic area, lateral septum,the head of caudate-putamen and cingulate cortex. No binding wasobserved in cortex, nucleus accumbens, hippocampus (except inpresubiculum) or cerebellum. The localization of NPFF binding sites isin good agreement with the location of the peptide itself, consistentwith the binding sites mediating the biological actions of NPFF in thesetissues (29, 34, 35). Less is known about the signal transductionpathways activated by NPFF receptors; NPFF was shown to activateadenylyl cyclase in mouse olfactory bulb membranes (36) but no otherreports of functional coupling via G proteins have appeared.

Until now, no direct evidence for NPFF receptor subtypes has beenreported in mammals. Recent physiological data suggest complex(biphasic) effects on nociception and antiopiate activity of NPFF (forreview, see (3, 4)) that could possibly signal the presence of multiplesubtypes. Short term ICV injection of NPFF causes a hyperesthesic effectfollowed by long lasting analgesic effect. Intrathecal NPFF and FMRFaboth produce long-lasting analgesia, but subeffective doses causeddifferent modulatory effects on morphine-induced analgesia (F8Fapotentiated, FMRFa decreased). The analgesic effects of NPFF aresensitive to naloxone, suggesting that NPFF receptors may have distinctpresynaptic (possibly associated with increase release of opioids) andpostsynaptic (anti-opiate) effects mediated by multiple receptors.Little is known of the biological effects of A18Famide, which shares itsC-terminal 4 amino acids with NPFF, but the existence of a family ofrelated peptides often is predictive of multiple receptor subtypes.

No nonpeptide agonists or antagonists of NPFF are available, but severaluseful peptidic analogs have been developed that exhibit increasedagonist stability or antagonist activity. For example, desamino Y8Fa(daY8Fa) can antagonize the behavioral effects of NPFF and restoremorphine-sensitivity (tail-flick analgesia) to morphine-tolerant rats atlower doses, although at higher doses it can act as NPFF agonist(10)(see also (3)). (1DMe)Y8Fa, in which L-Phe¹ is replaced by D-Tyr andthe second peptidic bond is N-methylated, has been shown to inhibitmorphine-induced analgesia (18), and has higher affinity and stabilitythan Y8Fa: (1DMe)Y8Fa was 90% stable after 150 min. incubation with ratspinal cord membranes compared with Y8Fa, which was fully degraded after30 minutes. These analogs may be useful in predicting the effects ofagonist or antagonist drugs that would act at NPFF receptors.

Despite the numerous studies linking NPFF with analgesia (for review,see (4)), only recently has NPFF been observed to play a role in animalmodels of chronic pain. For example, NPFF has recently been shown to beinvolved in inflammatory pain (37) and neuropathic pain (38).Importantly, NPFF was shown to attenuate the allodynia associated withneuropathic pain, suggesting that it may be clinically useful intreating this condition. In addition to its potential therapeutic rolesin the treatment of pain and morphine tolerance ((4) and above), NPFFand related peptides have a number of other biological activities thatmay be therapeutically relevant. NPFF and FMRFamide have been shown toreduce deprivation- and morphine-induced feeding in rats (39-41),indicating that NPFF receptors may be important targets in the treatmentof eating disorders. FMRFamide has also been shown to produceantipsychotic (42) and antianxiety (85) effects in rats, indicating thatNPFF receptors may be valuable targets for the treatment of psychosisand anxiety. There is evidence for a role of NPFF in learning andmemory. Kavaliers and Colwell (79) have shown that i.c.v. administeredNPFF has a biphasic effect of spatial learning in mice: low dosesimprove and high doses impair learning. This suggests the possibilitythat different NPFF receptor subtypes may have opposite roles in sometypes of learning behavior. NPFF is known to have indirect effects onwater and electrolyte balance. Arima et al. (86) have shown that NPFFwill reduce increase in vasopressin release produced by salt loading orhypovolemia. Additionally, NPFF may be involved in the control of plasmaaldosterone levels (87). These observations raise the possibility thatagents targeting NPFF receptors may be of value in the treatment ofdiuresis or in the treatment of cardiovascular conditions such ashypertension and congestive heart failure. Drugs acting at NPFFreceptors may be of value in the treatment of diabetes, since NPFF andA-18-Famide have been shown to produce significant inhibition ofglucose- and arginine-induced insulin release in rats (88). Severalinvestigators have reported effects of NPFF and analogs on intestinalmotility in mice (89) and guinea pigs (90, 91). When administered toisolated preparations of guinea pig ileum, the actions of NPFF opposethose of opioids. Conversely, i.c.v. administration of NPFF in miceproduces effects similar to those of morphine on intestinal motility.Together, these results indicate a complex modulatory role for NPFF inintestinal motility, but indicate that NPFF receptors are potentialtargets for drugs to treat GI motility disorders, including irritablebowel syndrome. NPFF has been shown to precipitate nicotine abstinencesyndrome in a rodent model (43). These authors have raised thepossibility that nicotine dependence may be attenuated by measures whichinactivate NPFF. Thus, NPFF receptor antagonists may be of use for thispurpose. Finally, NPFF is known to elicit two acute cardiovascularresponses when administered peripherally: elevation of blood pressureand heart rate (24, 25). These actions may be mediated peripherally,centrally, or both. Thus, agents acting at NPFF receptors may be ofvalue in the treatment of hypertension (also see above) or hypotension.The cloning of NPFF receptors will facilitate the elucidation of theroles of NPFF and related peptides in these and other importantbiological functions.

Described herein is the isolation and characterization of a new familyof neuropeptide FF (NPFF) receptors, referred to herein as the NPFFreceptors. Cloned NPFF receptors will serve as invaluable tools for drugdesign for pathophysiological conditions such as memory loss, affectivedisorders, schizophrenia, pain, hypertension, locomotor problems,circadian rhythm disorders, eating/body weight disorders,sexual/reproductive disorders, nasal congestion, diarrhea,gastrointestinal, and cardiovascular disorders. Also described hereinare experimental data which indicate that NPFF receptors will be usefultargets for the design of drugs to treat disorders of the lower urinarytract, including incontinence and bladder instability.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid encoding a mammalianNPFF receptor.

This invention provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in SEQ ID NO:1 under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a NPFF peptide is added to the culture and the CHO cellsexpress the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. This invention further provides anucleic acid encoding a mammalian NPFF receptor, wherein the nucleicacid (a) hybridizes to a nucleic acid having the defined sequence shownin SEQ ID NO:3 under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a NPFF peptideis added to the culture and the CHO cells express the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement. This invention also provides a nucleic acid encoding amammalian NPFF receptor, wherein the nucleic acid (a) hybridizes to anucleic acid having the defined sequence shown in SEQ ID NO:5 under lowstringency conditions or a sequence complementary thereto and (b) isfurther characterized by its ability to cause a change in the pH of aculture of CHO cells when a NPFF peptide is added to the culture and theCHO cells express the nucleic acid which hybridized to the nucleic acidhaving the defined sequence or its complement.

This invention further provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in SEQ ID NO:7 under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a NPFF peptide is added to the culture and the CHO cellsexpress the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement.

This invention further provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in SEQ ID NO:43 under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a NPFF peptide is added to the culture and the CHO cellsexpress the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement.

This invention also provides a purified mammalian NPFF receptor protein.

This invention further provides a vector comprising a nucleic acidencoding a mammalian NPFF receptor, particularly a vector adapted forexpression of the mammalian NPFF receptor in mammalian or non-mammaliancells.

This invention provides a plasmid designated pEXJ-rNPFF1 (ATCC AccessionNo. 203184). This invention also provides a plasmid designatedpWE15-hNPFF1 (ATCC Accession No. 203183). This invention furtherprovides a plasmid designated pcDNA3.1-hNPFF2b (ATCC Accession No.203255). This invention still further provides a plasmid designatedpcDNA3.1-hNPFF1 (ATCC Accession No. 203605). This invention stillfurther provides a plasmid designated pcDNA3.1-rNPFF2-f (Patent DepositDesignation No. PTA-535).

This invention additionally provides a cell comprising a vector which inturn comprises a nucleic acid encoding a mammalian NPFF receptor as wellas a membrane preparation isolated from such a cell.

Moreover, this invention provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within one of the twostrands of the nucleic acid encoding the mammalian NPFF1 receptor andcontained in plasmid pEXJ-rNPFF1 (ATCC Accession No. 203184), plasmidpWE15-hNPFF1 (ATCC Accession No. 203183), plasmid pCDNA3.1-hNPFF2b (ATCCAccession No. 203255), plasmid pcDNA3.1-hNPFF1 (ATCC Accession No.203605) or plasmid pcDNA3.1-rNPFF2-f (Patent Deposit Designation No.PTA-535).

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:1 or (b) the reverse complement thereto.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:3 or (b) the reverse complement thereto.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:5 or (b) the reverse complement thereto.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:7 or (b) the reverse complement thereto.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:43 or (b) the reverse complement thereto.

This invention still further provides an antisense oligonucleotidehaving a sequence capable of specifically hybridizing to RNA encodingthe mammalian NPFF receptor, so as to prevent translation of the RNA.This invention also provides an antisense oligonucleotide having asequence capable of specifically hybridizing to genomic DNA encoding amammalian NPFF receptor, so as to prevent transcription thereof.

This invention further provides an antibody capable of binding to amammalian NPFF receptor. This invention also provides an agent capableof competitively inhibiting the binding of the antibody to a mammalianNPFF receptor.

In addition, this invention provides a pharmaceutical compositioncomprising (a) an amount of the oligonucleotide described above capableof passing through a cell membrane and effective to reduce expression ofa mammalian NPFF receptor and (b) a pharmaceutically acceptable carriercapable of passing through the cell membrane.

This invention also provides a transgenic, nonhuman mammal expressingDNA encoding a mammalian NPFF receptor. This invention also provides atransgenic, nonhuman mammal comprising a homologous recombinationknockout of the native mammalian NPFF receptor. This invention furtherprovides a transgenic, nonhuman mammal whose genome comprises antisenseDNA complementary to the DNA encoding a mammalian NPFF receptor soplaced within the genome as to be transcribed into antisense mRNA whichis complementary to mRNA encoding the mammalian NPFF receptor and whichhybridizes to mRNA encoding the mammalian NPFF receptor, therebyreducing its translation.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian NPFF receptor which comprisescontacting cells containing DNA encoding and expressing on their cellsurface the mammalian NPFF receptor, wherein such cells do not normallyexpress the mammalian NPFF receptor, with the compound under conditionssuitable for binding, and detecting specific binding of the chemicalcompound to the mammalian NPFF receptor.

This invention further provides a process for identifying a chemicalcompound which specifically binds to a mammalian NPFF receptor whichcomprises contacting a membrane preparation from cells transfected withDNA encoding and expressing on their cell surface the mammalian NPFFreceptor, wherein such cells do not normally express the mammalian NPFFreceptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the mammalianNPFF receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianNPFF receptor which comprises separately contacting cells expressing ontheir cell surface the mammalian NPFF receptor, wherein such cells donot normally express the mammalian NPFF receptor, with both the chemicalcompound and a second chemical compound known to bind to the receptor,and with only the second chemical compound, under conditions suitablefor binding of both compounds, and detecting specific binding of thechemical compound to the mammalian NPFF receptor, a decrease in thebinding of the second chemical compound to the mammalian NPFF receptorin the presence of the chemical compound indicating that the chemicalcompound binds to the mammalian NPFF receptor.

This invention further provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian NPFF receptor which comprises separately contacting a membranefraction from cells expressing on their cell surface the mammalian NPFFreceptor, wherein such cells do not normally express the mammalian NPFFreceptor, with both the chemical compound and a second chemical compoundknown to bind to the receptor, and with only the second chemicalcompound, under conditions suitable for binding of both compounds, anddetecting specific binding of the chemical compound to the mammalianNPFF receptor, a decrease in the binding of the second chemical compoundto the mammalian NPFF receptor in the presence of the chemical compoundindicating that the chemical compound binds to the mammalian NPFFreceptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian NPFF receptor to identify acompound which specifically binds to the mammalian NPFF receptor, whichcomprises (a) contacting cells transfected with and expressing DNAencoding the mammalian NPFF receptor with a compound known to bindspecifically to the mammalian NPFF receptor; (b) contacting thepreparation of step (a) with the plurality of compounds not known tobind specifically to the mammalian NPFF receptor, under conditionspermitting binding of compounds known to bind to the mammalian NPFFreceptor; (c) determining whether the binding of the compound known tobind to the mammalian NPFF receptor is reduced in the presence of anycompound within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.

This invention also provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian NPFF receptor toidentify a compound which specifically binds to the mammalian NPFFreceptor, which comprises (a) contacting a membrane preparation fromcells transfected with and expressing DNA encoding a mammalian NPFFreceptor with a compound known to bind to the mammalian NPFF receptor;(b) determining whether the binding of a compound known to bind to themammalian NPFF receptor is reduced in the presence of any compoundwithin the plurality of compounds, relative to the binding of thecompound in the absence of the plurality of compounds; and if so (c)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.

Still further, this invention provides a method of detecting expressionof a mammalian NPFF receptor by detecting the presence of mRNA codingfor the mammalian NPFF receptor which comprises obtaining total mRNAfrom the cell and contacting the mRNA so obtained with a nucleic acidprobe under hybridizing conditions, detecting the presence of mRNAhybridizing to the probe, and thereby detecting the expression of themammalian NPFF receptor by the cell.

This invention provides a method of detecting the presence of amammalian NPFF receptor on the surface of a cell which comprisescontacting the cell with an antibody under conditions permitting bindingof the antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of the mammalianNPFF receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian NPFF receptors whichcomprises producing a transgenic, nonhuman mammal whose levels ofmammalian NPFF receptor activity are varied by use of an induciblepromoter which regulates mammalian NPFF receptor expression.

This invention also provides a method of determining the physiologicaleffects of varying levels of activity of mammalian NPFF receptors whichcomprises producing a panel of transgenic, nonhuman mammals eachexpressing a different amount of mammalian NPFF receptor.

This invention further provides a method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a mammalian NPFF receptorcomprising administering a compound to a transgenic, nonhuman mammal asdescribed above and determining whether the compound alleviates thephysical and behavioral abnormalities displayed by the transgenic,nonhuman mammal as a result of overactivity of a mammalian NPFFreceptor, the alleviation of the abnormality identifying the compound asan antagonist. This invention also provides an antagonist identified bythis method. This invention still further provides a pharmaceuticalcomposition comprising an antagonist identified by this method and apharmaceutically acceptable carrier.

This invention additionally provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by decreasing theactivity of a mammalian NPFF receptor which comprises administering tothe subject an effective amount of the preceding pharmaceuticalcomposition containing a mammalian NPFF receptor antagonist, therebytreating the abnormality.

This invention also provides a method for identifying an agonist capableof alleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a mammalian NPFF receptorcomprising administering a compound to a transgenic, nonhuman mammal,and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal,the alleviation of the abnormality identifying the compound as anagonist. This invention also provides an agonist identified by thismethod. This invention further provides a pharmaceutical compositioncomprising an agonist identified by this method and a pharmaceuticallyacceptable carrier. This invention provides a method of treating anabnormality in a subject wherein the abnormality is alleviated byincreasing the activity of a mammalian NPFF receptor which comprisesadministering to the subject an effective amount of the precedingpharmaceutical composition containing a mammalian NPFF receptor agonist,thereby treating the abnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific mammalian allelewhich comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a mammalian NPFF receptor and labeled with a detectable marker;(e) detecting labeled bands which have hybridized to the DNA encoding amammalian NPFF receptor labeled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and(g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step (e) and the DNA obtained fordiagnosis from step (f) to determine whether the patterns are the sameor different and to diagnose thereby predisposition to the disorder ifthe patterns are the same.

This invention provides a method of preparing a purified mammalian NPFFreceptor which comprises: (a) culturing cells which express themammalian NPFF receptor; (b) recovering the mammalian NPFF receptor fromthe cells; and (c) purifying the mammalian NPFF receptor so recovered.

This invention provides a method of preparing a purified mammalian NPFFreceptor which comprises: (a) inserting a nucleic acid encoding themammalian NPFF receptor into a suitable vector; (b) introducing theresulting vector into a suitable host cell; (c) placing the resultingcell in suitable condition permitting the production of the mammalianNPFF receptor; (d) recovering the mammalian NPFF receptor produced bythe resulting cell; and (e) isolating and/or purifying the mammalianNPFF receptor so recovered.

This invention provides a process for determining whether a chemicalcompound is a mammalian NPFF receptor agonist which comprises contactingcells transfected with and expressing DNA encoding the mammalian NPFFreceptor with the compound under conditions permitting the activation ofthe mammalian NPFF receptor, and detecting an increase in mammalian NPFFreceptor activity, so as to thereby determine whether the compound is amammalian NPFF receptor agonist. This invention also provides apharmaceutical composition which comprises an amount of a mammalian NPFFreceptor agonist determined by this process effective to increaseactivity of a mammalian NPFF receptor and a pharmaceutically acceptablecarrier.

This invention provides a process for determining whether a chemicalcompound is a mammalian NPFF receptor antagonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian NPFF receptor with the compound in the presence of a knownmammalian NPFF receptor agonist, under conditions permitting theactivation of the mammalian NPFF receptor, and detecting a decrease inmammalian NPFF receptor activity, so as to thereby determine whether thecompound is a mammalian NPFF receptor antagonist. This invention alsoprovides a pharmaceutical composition which comprises an amount of amammalian NPFF receptor antagonist determined by this process effectiveto reduce activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

This invention provides a process for determining whether a chemicalcompound specifically binds to and activates a mammalian NPFF receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the mammalian NPFF receptor,wherein such cells do not normally express the mammalian NPFF receptor,with the chemical compound under conditions suitable for activation ofthe mammalian NPFF receptor, and measuring the second messenger responsein the presence and in the absence of the chemical compound, a change inthe second messenger response in the presence of the chemical compoundindicating that the compound activates the mammalian NPFF receptor. Thisinvention also provides a compound determined by this process. Thisinvention further provides a pharmaceutical composition which comprisesan amount of the compound (a NPFF receptor agonist) determined by thisprocess effective to increase activity of a mammalian NPFF receptor anda pharmaceutically acceptable carrier.

This invention provides a process for determining whether a chemicalcompound specifically binds to and inhibits activation of a mammalianNPFF receptor, which comprises separately contacting cells producing asecond messenger response and expressing on their cell surface themammalian NPFF receptor, wherein such cells do not normally express themammalian NPFF receptor, with both the chemical compound and a secondchemical compound known to activate the mammalian NPFF receptor, andwith only the second chemical compound, under conditions suitable foractivation of the mammalian NPFF receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change in the second messenger response inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compoundindicating that the chemical compound inhibits activation of themammalian NPFF receptor. This invention also provides a compounddetermined by this process. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian NPFF receptor antagonist) determined by this effective toreduce activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a mammalian NPFF receptor to identify acompound which activates the mammalian NPFF receptor which comprises:(a) contacting cells transfected with and expressing the mammalian NPFFreceptor with the plurality of compounds not known to activate themammalian NPFF receptor, under conditions permitting activation of themammalian NPFF receptor; (b) determining whether the activity of themammalian NPFF receptor is increased in the presence of the compounds;and if so (c) separately determining whether the activation of themammalian NPFF receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the mammalian NPFF receptor. This invention also provides acompound identified by this method. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian NPFF receptor agonist) identified by this method effective toincrease activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian NPFFreceptor to identify a compound which inhibits the activation of themammalian NPFF receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian NPFF receptor with theplurality of compounds in the presence of a known mammalian NPFFreceptor agonist, under conditions permitting activation of themammalian NPFF receptor; (b) determining whether the activation of themammalian NPFF receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian NPFF receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian NPFF receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian NPFF receptor. This invention also provides a compoundidentified by this method. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian NPFF receptor antagonist) identified by this process effectiveto decrease activity of a mammalian NPFF receptor and a pharmaceuticallyacceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian NPFF receptor which comprises administering to the subject anamount of a compound which is a mammalian NPFF receptor agonisteffective to treat the abnormality.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of amammalian NPFF receptor which comprises administering to the subject anamount of a compound which is a mammalian NPFF receptor antagonisteffective to treat the abnormality.

This invention provides a process for making a composition of matterwhich specifically binds to a mammalian NPFF receptor which comprisesidentifying a chemical compound using any of the processes describedherein for identifying a compound which binds to and/or activates orinhibits activation of a mammalian NPFF receptor and then synthesizingthe chemical compound or a novel structural and functional analog orhomolog thereof. This invention further provides a process for preparinga pharmaceutical composition which comprises admixing a pharmaceuticallyacceptable carrier and a pharmaceutically acceptable amount of achemical compound identified by any of the processes described hereinfor identifying a compound which binds to and/or activates or inhibitsactivation of a mammalian NPFF receptor or a novel structural andfunctional analog or homolog thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Nucleotide sequence encoding a rat neuropeptide FF receptor (NPFF1)(SEQID NO: 1). In addition, partial 5′ and 3′ untranslated sequences areshown. In FIG. 1, two start (ATG) codons (at positions 73-75 and148-150) and the stop (TAG) codon (at positions 1369-1371) areunderlined.

FIG. 2

Deduced amino acid sequence for rat NPFF1 (SEQ ID NO: 2). Seven solidlines designated I-VII located above portions of the sequence indicatethe seven putative transmembrane (TM) spanning regions.

FIG. 3

Partial amino acid alignment of rat and human NPFF1. Vertical linesrepresent identical residues and dots represent similar residues.

FIG. 4

Nucleotide sequence of hNPFF2b (SEQ ID NO: 5). The initiating methionineand the stop codon are underlined.

FIG. 5

Deduced amino acid sequence for human hNPFF2 (SEQ ID NO: 6), withpotential transmembrane domains underlined.

FIG. 6

Amino acid alignment of rat NPFF1 and human NPFF2. Vertical linesrepresent identical residues and dots represent similar residues.

Figure Legends

FIG. 7

Nucleotide sequence of a human neuropeptide FF receptor (NPFF1) (SEQ IDNO: 7). The initiating methionine (at positions 1-3) and the stop codon(at positions 1291-1293) are underlined.

FIG. 8

Deduced amino acid sequence for human NPFF1 (SEQ ID NO: 8). Seven solidlines designated I-VII indicate the seven putative transmembrane (TM)spanning regions.

FIG. 9

Amino acid alignment of the human NPFF1 and human NPFF2 receptors.Vertical lines represent identical residues and dots represent similarresidues.

FIGS. 10A-10C

Electrophysiological responses to NPFF and related peptides from voltageclamped oocytes expressing NPFF1 and chimeric G-protein.

FIGS. 11A-11C

Electrophysiological responses in voltage-clamped oocytes expressingNPFF2 mRNA. FIG. 11A: Oocyte injected with NPFF2 mRNA (from ligationPCR) generates an inward current in response to NPFF at 1 μM. FIG. 11B:In a different oocyte, no response is observed when challenged with amixture of galanin, NPY, orexin A and neurokinin A, each at 1 μM. Asubsequent application of NPFF elicits a response. FIG. 11C: Oocyteinjected with NPFF2 mRNA (from B089) generates an inward current inresponse to NPFF at 1 μM. Oocytes were clamped at a holding potential of−80 mV.

FIGS. 12A and 12B

Microphysiometric response of CHO cells transiently transfected witheither NPFF1 (SN2) alone or NPFF1 accompanied by Gq/Gz. FIG. 12A: Cellsexpressing either NPFF1 alone or NPFF1+Gq/Gz produced a dose-dependentresponse to NPFF with an EC50 value of 19.3 nM and 27.7 nM respectively.Mock control cells transfected with empty vector produced little if anyresponse to NPFF even at the highest concentrations used. FIG. 12B:Cells expressing NPFF1 alone produced a dose-dependent response toA-18-F-amide with an EC50 value of 150 nM. In both FIGS. 12A and 12Bcontrol cells mock transfected with empty vector produced little if anyresponse to drug even at the highest concentrations used. Responses arereported as percentage increase in the acidification rate as observedjust prior to drug challenge (immediate prior basal rate).

FIGS. 13A and 13B

NPFF stimulation of Inositol phosphate release in NPFF-1 transfectedCos-7 cells. FIG. 13A: Cos-7 cells were transiently transfected withNPFF-1 receptor cDNA. FIG. 13B: Cos-7 cells were transientlyco-transfected with cDNAs for the NPFF-1 receptor and the Gq/Gz chimera.The accumulation of total inositol phosphate release was measured byprelabelling cells with [³H]myoinositol (2 μCi/ml) overnight. Cells werewashed to remove unincorporated radioactivity and resuspended in mediumcontaining 10 mM LiCl. [³H]myoinositol labeled cells were incubated withappropriate drugs for 1 hr at 37° C. The reaction was stopped byaddition of 5% TCA and IPs were isolated by ion exchange chromatography(Berridge et al., 1982). Columns were washed with water and total [³H]inositol phosphates were then eluted with 1M ammonium formate/0.1 Mformic acid. Radioactivity in the final fraction was measured by liquidscintillation spectroscopy. Cells were either treated with vehicle(water, control) or cholera toxin (CTX; 1 μg/ml) or pertussis toxin(PTX, 100 ng/ml) overnight. Data are from one experiment representativeof at least one other.

FIG. 14

RT-PCR was performed as described on a panel of mRNA extracted from rattissue as indicated at the bottom of the gel. After amplification, PCRreactions were size fractionated on 10% polyacrylamide gels, and stainedwith SYBR Green I. Images were analyzed using a Molecular Dynamics Storm860 workstation. The amplified band corresponding to NPFF1 (490 basepairs) is indicated (arrow). RT-PCR indicates a broad distribution ofmRNA encoding NPFF1 with highest concentrations found in nervous systemstructures.

FIG. 15

Autoradiograph demonstrating hybridization of radiolabeled rat NPFF1probe to RNA extracted from rat tissue in a solutionhybridization/nuclease protection assay using ³²P labeled riboprobe. 2μg of RNA was used in each assay. The single band (arrow) representsmRNA coding for the NPFF1 receptors extracted from the indicated tissue.Highest levels of mRNA coding for NPFF1 are found in: hypothalamus andpituitary gland. The smaller bands representing NPFF1 mRNA from thepituitary, adrenal gland, and ovary (double arrow) may indicate a splicevariant present in this tissue. Integrity of RNA was assessed usinghybridization to mRNA coding for GAPDH (not shown).

FIG. 16

RT-PCR was performed as described on a panel of mRNA extracted fromtissue as indicated at the bottom of the gel. After amplification, PCRreactions were size fractionated on 10% polyacrylamide gels, and stainedwith SYBR Green I. Images were analyzed using a Molecular Dynamics Storm860 workstation. The amplified band corresponding to NPFF2 receptors(approximately 325 base pairs) is indicated (arrow). RT-PCR indicates abroad distribution of mRNA encoding NPFF2 receptors. The only tissuecontaining mRNA coding for NPFF2 receptors were HeLa cells and Jurkatcells.

FIGS. 17A-17C

Nucleotide sequence encoding a rat neuropeptide FF receptor (NPFF2). Inaddition, partial 5′ and 3′ untranslated sequences are shown. Two start(ATG) codons (at positions 26-28 and 128-130) and the stop (TAG) codon(at positions 1277-1279) are underlined.

FIGS. 18A and 18B

Deduced amino acid sequence of the rat neuropeptide FF receptor (NPFF2)encoded by the nucleotide sequence shown in FIGS. 17A-17C. Sevenputative transmembrane spanning regions are indicated by underlining.

FIGS. 19A and 19B

Amino acid alignment of human NPFF2 and rat NPFF2. Vertical linesrepresent identical residues and dots represent similar residues.

FIGS. 20A and 20B

Amino acid alignment of rat NPFF1 and rat NPFF2. Vertical linesrepresent identical residues and dots represent similar residues.

FIG. 21

Inhibition of distension-induced rhythmic contractions of the bladder inan anesthetized rat by NPFF (1.0 mg/kg) administered intravenously.

FIG. 22

Effect of saline, frog pancreatic polypeptide (fPP), and increasingconcentrations of NPFF on the disappearance time of the bladdercontractions in the distension-induced rhythmic contraction model ofmicturition in anesthetized rats. Presented are the mean values ±semfrom experiments on “n” different rats.

FIG. 23

Inhibition of distension-induced rhythmic contractions of the bladder inan anesthetized rat by frog Pancreatic Polypeptide (fPP) (0.3 mg/kg)administered intravenously.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

-   -   A=adenine    -   G=guanine    -   C=cytosine    -   T=thymine    -   U=uracil    -   M=adenine or cytosine    -   R=adenine or guanine    -   W=adenine, thymine, or uracil    -   S=cytosine or guanine    -   Y=cytosine, thymine, or uracil    -   K=guanine, thymine, or uracil    -   V=adenine, cytosine, or guanine (not thymine or uracil    -   H=adenine, cytosine, thymine, or uracil (not guanine)    -   D=adenine, guanine, thymine, or uracil (not cytosine)    -   B=cytosine, guanine, thymine, or uracil (not adenine)    -   N=adenine, cytosine, guanine, thymine, or uracil (or other        modified base such as inosine)    -   I=inosine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the polypeptides of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which decreases the activity of any of thepolypeptides of the subject invention.

The activity of a G-protein coupled receptor such as the polypeptidesdisclosed herein may be measured using any of a variety of functionalassays in which activation of the receptor in question results in anobservable change in the level of some second messenger system,including, but not limited to, adenylate cyclase, calcium mobilization,arachidonic acid release, ion channel activity, inositol phospholipidhydrolysis or guanylyl cyclase. Heterologous expression systemsutilizing appropriate host cells to express the nucleic acid of thesubject invention are used to obtain the desired second messengercoupling. Receptor activity may also be assayed in an oocyte expressionsystem.

It is possible that the mammalian NPFF receptor genes contain intronsand furthermore, the possibility exists that additional introns couldexist in coding or non-coding regions. In addition, spliced form(s) ofmRNA may encode additional amino acids either upstream of the currentlydefined starting methionine or within the coding region. Further, theexistence and use of alternative exons is possible, whereby the mRNA mayencode different amino acids within the region comprising the exon. Inaddition, single amino acid substitutions may arise via the mechanism ofRNA editing such that the amino acid sequence of the expressed proteinis different than that encoded by the original gene. (Burns et al., 1996(82); Chu et al., 1996 (83)). Such variants may exhibit pharmacologicproperties differing from the polypeptide encoded by the original gene.

This invention provides splice variants of the mammalian NPFF receptorsdisclosed herein. This invention further provides for alternatetranslation initiation sites and alternately spliced or edited variantsof nucleic acids encoding the mammalian NPFF receptors of thisinvention.

The nucleic acids of the subject invention also include nucleic acidanalogs of the rat and human NPFF receptor genes, wherein the rat NPFF1receptor gene comprises the nucleic acid sequence shown in SEQ ID NO:1or contained in plasmid pEXJ-rNPFF1 (ATCC Accession No. 203184); thehuman NPFF1 receptor gene comprises the nucleic acid shown in SEQ IDNO:3 and contained in plasmid pWE15-hNPFF1 (ATCC Accession No. 203183);the human NPFF2 receptor gene comprises the nucleic acid shown in SEQ IDNO:5 and contained in plasmid pcDNA3.1-hNPFF2b (ATCC Accession No.203255); the human NPFF1 receptor gene comprises the nucleic acid shownin SEQ ID NO:7 and contained in plasmid pcDNA3.1-hNPFF1 (ATCC AccessionNo. 203605); or the rat NPFF2 receptor gene comprises the nucleic acidshown in SEQ ID NO:43 and contained in plasmid pcDNA3.1-rNPFF2-f (ATCCAccession No. Patent Deposit Designation No. PTA-535). Nucleic acidanalogs of the rat and human NPFF receptor genes differ from the rat andhuman NPFF receptor genes described herein in terms of the identity orlocation of one or more nucleic acid bases (deletion analogs containingless than all of the nucleic acid bases shown in SEQ ID NOs:1, 3, 5, 7or 43 or contained in plasmids pEXJ-rNPFF1, pWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1, or pcDNA3.1-rNPFF2-f respectively,substitution analogs wherein one or more nucleic acid bases shown in SEQID NOs:1, 3, 5, 7 or 43 or contained in plasmids pEXJ-rNPFF1,pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1, or pcDNA3.1-rNPFF2-frespectively, are replaced by other nucleic acid bases, and additionanalogs, wherein one or more nucleic acid bases are added to a terminalor medial portion of the nucleic acid sequence) and which encodeproteins which share some or all of the properties of the proteinsencoded by the nucleic acid sequences shown in SEQ ID NOs:1, 3, 5, 7 or43 or contained in plasmids pEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b,pcDNA3.1-hNPFF1, or pcDNA3.1-rNPFF2-f respectively. In one embodiment ofthe present invention, the nucleic acid analog encodes a protein whichhas an amino acid sequence identical to that shown in SEQ ID NO:2, 4, 6,8 or 44 or encoded by the nucleic acid sequence contained in plasmidspEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1, orpcDNA3.1-rNPFF2-f respectively. In another embodiment, the nucleic acidanalog encodes a protein having an amino acid sequence which differsfrom the amino acid sequences shown in SEQ ID NO:2, 4, 6, 8 or 44 orencoded by the nucleic acid contained in plasmids pEXJ-rNPFF1,pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 or pcDNA3.1-rNPFF2-frespectively. In a further embodiment, the protein encoded by thenucleic acid analog has a function which is the same as the function ofthe receptor proteins having the amino acid sequence shown in SEQ IDNO:2, 4, 6, 8 or 44. In another embodiment, the function of the proteinencoded by the nucleic acid analog differs from the function of thereceptor protein having the amino acid sequence shown in SEQ ID NO:2, 4,6, 8 or 44. In another embodiment, the variation in the nucleic acidsequence occurs within the transmembrane (TM) region of the protein. Ina further embodiment, the variation in the nucleic acid sequence occursoutside of the TM region.

This invention provides the above-described isolated nucleic acid;wherein the nucleic acid is DNA. In an embodiment, the DNA is cDNA. Inanother embodiment, the DNA is genomic DNA. In still another embodiment,the nucleic acid is RNA. Methods for production and manipulation ofnucleic acid molecules are well known in the art.

This invention further provides nucleic acid which is degenerate withrespect to the DNA encoding any of the polypeptides described herein. Inan embodiment, the nucleic acid comprises a nucleotide sequence which isdegenerate with respect to the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:43 or the nucleotidesequence contained in the plasmids pEXJ-rNPFF1, pWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1, or pcDNA3.1-rNPFF2-f respectively,that is, a nucleotide sequence which is translated into the same aminoacid sequence.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of the polypeptides of this invention,but which should not produce phenotypic changes. Alternately, thisinvention also encompasses DNAs, cDNAs, and RNAs which hybridize to theDNA, cDNA, and RNA of the subject invention. Hybridization methods arewell known to those of skill in the art.

The nucleic acids of the subject invention also include nucleic acidmolecules coding for polypeptide analogs, fragments or derivatives ofantigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs wherein one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors. Thecreation of polypeptide analogs is well known to those of skill in theart (R. F. Spurney et al. (1997); Fong, T. M. et al. (1995); Underwood,D. J. et al. (1994); Graziano, M. P. et al. (1996); Guam X. M. et al.(1995)).

The modified polypeptides of this invention may be transfected intocells either transiently or stably using methods well-known in the art,examples of which are disclosed herein. This invention also provides forbinding assays using the modified polypeptides, in which the polypeptideis expressed either transiently or in stable cell lines. This inventionfurther provides a compound identified using a modified polypeptide in abinding assay such as the binding assays described herein.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptides by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

This invention provides an isolated nucleic acid encoding a mammalianNPFF receptor. In one embodiment, the nucleic acid is DNA. In anotherembodiment, the DNA is cDNA. In another embodiment, the DNA is genomicDNA. In another embodiment, the nucleic acid is RNA.

In one embodiment, the mammalian NPFF receptor is a NPFF1 receptor. In afurther embodiment, the mammalian NPFF1 receptor is a rat NPFF1receptor. In another embodiment, the mammalian NPFF1 receptor is a humanNPFF1 receptor. In a further embodiment, the mammalian NPFF receptor isa NPFF2 receptor. In one embodiment, the mammalian NPFF2 receptor is ahuman NPFF2 receptor. In another embodiment, the mammalian NPFF2receptor is a rat NPFF2 receptor.

This invention also provides an isolated nucleic acid encoding specieshomologs of the NPFF receptors encoded by the nucleic acid sequenceshown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ IDNO:43 encoded by the plasmid pEXJ-rNPEF1, pWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1, or pcDNA3.1-rNPFF2-f respectively. Inone embodiment, the nucleic acid encodes a mammalian NPFF receptorhomolog which has substantially the same amino acid sequence as does theNPFF receptor encoded by the plasmid pEXJ-rNPFF1, pWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 or pcDNA3.1-rNPFF2-f. In anotherembodiment, the nucleic acid encodes a mammalian NPFF receptor homologwhich has above 65% amino acid identity to the NPFF receptor encoded bythe plasmid pEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1or pcDNA3.1-rNPFF2-f; preferably above 75% amino acid identity to theNPFF receptor encoded by the plasmid pEXJ-rNPFF1, pWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 or pcDNA3.1-rNPFF2-f; more preferablyabove 85% amino acid identity to the NPFF receptor encoded by theplasmid pEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 orpcDNA3.1-rNPFF2-f; most preferably above 95% amino acid identity to theNPFF receptor encoded by the plasmid pEXJ-rNPFF1, PWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1, or pcDNA3.1-rNPFF2-f. In anotherembodiment, the mammalian NPFF receptor homolog has above 70% nucleicacid identity to the NPFF receptor gene contained in plasmidpEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 orpcDNA3.1-rNPFF2-f; preferably above 80% nucleic acid identity to theNPFF receptor gene contained in the plasmid pEXJ-rNPFF1, pWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 or pcDNA3.1-rNPFF2-f; more preferablyabove 90% nucleic acid identity to the NPFF receptor gene contained inthe plasmid pEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1or pcDNA3.1-rNPFF2-f. Examples of methods for isolating and purifyingspecies homologs are described elsewhere (e.g., U.S. Pat. No. 5,602,024,WO94/14957, WO97/26853, WO98/15570).

In separate embodiments of the present invention, the nucleic acidencodes a NPFF receptor which has an amino acid sequence identical tothat encoded by the plasmid pEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b,pcDNA3.1-hNPFF1 or pcDNA3.1-rNPFF2-f. In further embodiments, the NPFFreceptor has a sequence substantially the same as the amino acidsequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 orSEQ ID NO:44. In other embodiments, the NPFF receptor has an amino acidsequence identical to the amino acid sequence shown in SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:44.

This invention provides an isolated nucleic acid encoding a modifiedmammalian NPFF receptor, which differs from a mammalian NPFF receptor byhaving an amino acid(s) deletion, replacement, or addition in the thirdintracellular domain.

This invention provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in SEQ ID NO:1 under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a NPFF peptide is added to the culture and the CHO cellsexpress the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. This invention further provides anucleic acid encoding a mammalian NPFF receptor, wherein the nucleicacid (a) hybridizes to a nucleic acid having the defined sequence shownin SEQ ID NO:3 under low stringency conditions or a sequencecomplementary thereto and (b) is further characterized by its ability tocause a change in the pH of a culture of CHO cells when a NPFF peptideis added to the culture and the CHO cells express the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement. This invention also provides a nucleic acid encoding amammalian NPFF receptor, wherein the nucleic acid (a) hybridizes to anucleic acid having the defined sequence shown in SEQ ID NO:5 under lowstringency conditions or a sequence complementary thereto and (b) isfurther characterized by its ability to cause a change in the pH of aculture of CHO cells when a NPFF peptide is added to the culture and theCHO cells express the nucleic acid which hybridized to the nucleic acidhaving the defined sequence or its complement.

This invention further provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in SEQ ID NO:7 under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a NPFF peptide is added to the culture and the CHO cellsexpress the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement.

This invention further provides a nucleic acid encoding a mammalian NPFFreceptor, wherein the nucleic acid (a) hybridizes to a nucleic acidhaving the defined sequence shown in SEQ ID NO:43 under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a NPFF peptide is added to the culture and the CHO cellsexpress the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement.

In one embodiment, the mammalian NPFF receptor is a rat NPFF1 receptor.In another embodiment, the mammalian NPFF receptor is a human NPFF1receptor. In a further embodiment, the mammalian NPFF receptor is ahuman NPFF2 receptor. In a further embodiment, the mammalian NPFFreceptor is a rat NPFF2 receptor. For purpose of the inventionhybridization under low stringency conditions means hybridizationperformed at 40° C. in a hybridization buffer containing 25% formamide,5×SCC, 7 mM Tris, 1× Denhardt's, 25 μl/ml salmon sperm DNA. Wash at 40°C. in 0.1×SCC, 0.1% SDS. Changes in pH are measured throughmicrophysiometric measurement of receptor mediated extracellularacidification rates. Because cellular metabolism is intricately involvedin a broad range of cellular events (including receptor activation ofmultiple messenger pathways), the use of microphysiometric measurementsof cell metabolism can in principle provide a generic assay of cellularactivity arising from the activation of any receptor regardless of thespecifics of the receptor's signaling pathway. General guidelines fortransient receptor expression, cell preparation and microphysiometricrecording are described elsewhere (Salon, J. A. and Owicki, J. A.,1996). Receptors and/or control vectors are transiently expressed inCHO-K1 cells, by liposome mediated transfection according to themanufacturers recommendations (LipofectAMINE, GibcoBRL, Gaithersburg,Md.), and maintained in Ham's F-12 complete (10% serum). A total of 10μg of DNA is used to transfect each 75 cm² flask which had been split 24hours prior to the transfection and judged to be 70-80% confluent at thetime of transfection. 24 hours post transfection, the cells areharvested and 3×10⁵ cells seeded into microphysiometer capsules. Cellsare allowed to attach to the capsule membrane for an additional 24hours; during the last 16 hours, the cells are switched to serum-freeF-12 complete to minimize ill-defined metabolic stimulation caused byassorted serum factors. On the day of the experiment the cell capsulesare transferred to the microphysiometer and allowed to equilibrate inrecording media (low buffer RPMI 1640, no bicarbonate, no serum(Molecular Devices Corporation, Sunnyvale, Calif.) containing 0.1% fattyacid free BSA), during which a baseline measurement of basal metabolicactivity is established. A standard recording protocol specifies a 100μl/min flow rate, with a 2 min total pump cycle which includes a 30 secflow interruption during which the acidification rate measurement istaken. Ligand challenges involve a 1 min 20 sec exposure to the samplejust prior to the first post challenge rate measurement being taken,followed by two additional pump cycles for a total of 5 min 20 secsample exposure. Typically, drugs in a primary screen are presented tothe cells at 10 μM final concentration. Ligand samples are then washedout and the acidification rates reported are expressed as a percentageincrease of the peak response over the baseline rate observed just priorto challenge. Endogenous NPFF peptides include rat NPFF (FLFQPQRF-NH2)(SEQ ID NO: 45) and rat A18Fa (AGEGLSSPFWSLAAPQRF-NH2) (SEQ ID NO: 46).

This invention provides a purified mammalian NPFF receptor protein. Inone embodiment, the purified mammalian NPFF receptor protein is a humanNPFF1 receptor protein. In another embodiment, the purified mammalianNPFF receptor protein is a rat NPFF1 receptor protein. In a furtherembodiment, the purified mammalian NPFF receptor protein is a humanNPFF2 receptor protein. In a further embodiment, the purified mammalianNPFF receptor protein is a rat NPFF2 receptor protein.

This invention provides a vector comprising nucleic acid encoding amammalian NPFF receptor. In one embodiment, the mammalian NPFF receptorprotein is a NPFF1 receptor protein. In another embodiment of thepresent invention the mammalian NPFF receptor protein is a NPFF2receptor protein. In one embodiment, the mammalian NPFF receptor is arat NPFF1 receptor. In another embodiment, the mammalian NPFF receptoris a human NPFF1 receptor. In a further embodiment, the mammalian NPFFreceptor is a human NPFF2 receptor. In a further embodiment, thepurified mammalian NPFF receptor protein is a rat NPFF2 receptor.

In an embodiment, the vector is adapted for expression in a cell whichcomprises the regulatory elements necessary for expression of thenucleic acid in the cell operatively linked to the nucleic acid encodingthe mammalian NPFF receptor as to permit expression thereof. In separateembodiments, the cell is a bacterial cell, an amphibian cell, a yeastcell, an insect cell or a mammalian cell. In another embodiment, thevector is a baculovirus. In one embodiment, the vector is a plasmid.

This invention provides a plasmid designated pEXJ-rNPFF1 (ATCC AccessionNo. 203184). This plasmid comprises the regulatory elements necessaryfor expression of DNA in a mammalian cell operatively linked to DNAencoding the mammalian NPFF1 receptor so as to permit expressionthereof. This invention also provides a plasmid designated pWE15-hNPFF1(ATCC. Accession No. 203183). This invention further provides a plasmiddesignated pcDNA3.1-hNPFF2b (ATCC Accession No. 203255). This inventionadditionally provides a plasmid designated pcDNA3.1-hNPFF1 (ATCCAccession No. 203605). This invention additionally provides a plasmiddesignated pcDNA3.1-rNPFF2-f (Patent Deposit Designation No. PTA-535).

These plasmids (pEXJ-rNPFF1 and pWE15-hNPFF1) were deposited on Sep. 9,1998, with the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procure and were accorded ATCCAccession Nos. 203184, and 203183, respectively. PlasmidpcDNA3.1-hNPFF2b was deposited on Sep. 22, 1998, with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. 20355.Plasmid pcDNA3.1-hNPFF1 was deposited on Jan. 21, 1999, with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Accession No. 203605. Plasmid pcDNA3.1-rNPFF2-f was deposited onAug. 17, 1999, with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, U.S.A. under the provisionsof the Budapest Treaty for the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure and was accordedPatent Deposit Designation No. PTA-535.

This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the polypeptidedepending upon the host cell used. In an embodiment, the vector orplasmid comprises the coding sequence of the polypeptide and theregulatory elements necessary for expression in the host cell.

This invention provides a cell comprising a vector comprising a nucleicacid encoding the mammalian NPFF receptor. In an embodiment, the cell isa non-mammalian cell. In a further embodiment, the non-mammalian cell isa Xenopus oocyte, cell or a Xenopus melanophore cell. In anotherembodiment, the cell is a mammalian cell. In a further embodiment, themammalian cell is a COS-7 cell, a 293 human embryonic kidney cell(HEK-293 cell), a NIH-3T3 cell, a LM(tk−) cell, a mouse Y1 cell, or aCHO cell.

This invention provides an insect cell comprising a vector adapted forexpression in an insect cell which comprises a nucleic acid encoding amammalian NPFF receptor. In another embodiment, the insect cell is anSf9 cell, an Sf21 cell or a Trichoplusia ni 5B1-4 (HighFive) cell.

This invention provides a membrane preparation isolated from any one ofthe cells described above.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within one of the twostrands of the nucleic acid encoding the mammalian NPFF receptor and arecontained in plasmid pEXJ-rNPFF1, plasmid pWE15-hNPFF1,pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 or pcDNA3.1-rNPFF2-f. This inventionalso provides a nucleic acid probe comprising at least 15 nucleotides,which probe specifically hybridizes with a nucleic acid encoding amammalian NPFF receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in SEQ ID NO:1 or (b) the reverse complement thereto. Thisinvention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:3 or (b) the reverse complement thereto.This invention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:5 or (b) the reverse complement thereto.This invention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:7 or (b) the reverse complement thereto.This invention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian NPFF receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in SEQ ID NO:43 or (b) the reverse complement thereto. Inone embodiment, the nucleic acid is DNA. In another embodiment, thenucleic acid is RNA.

As used herein, the phrase “specifically hybridizing” means the abilityof a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

Nucleic acid probe technology is well known to those skilled in the artwho will readily appreciate that such probes may vary greatly in lengthand may be labeled with a detectable label, such as a radioisotope orflourescent dye, to facilitate detection of the probe. DNA probemolecules may be produced by insertion of a DNA molecule which encodesthe polypeptides of this invention into suitable vectors, such asplasmids or bacteriophages, followed by transforming into suitablebacterial host cells, replication in the transformed bacterial hostcells and harvesting of the DNA probes, using methods well known in theart. Alternatively, probes may be generated chemically from DNAsynthesizers.

RNA probes may be generated by inserting the DNA molecule which encodesthe polypeptides of this invention downstream of a bacteriophagepromoter such as T3, T7, or SP6. Large amounts of RNA probe may beproduced by incubating the labeled nucleotides with the linearizedfragment where it contains an upstream promoter in the presence of theappropriate RNA polymerase.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to RNA encoding a mammalian NPFFreceptor, so as to prevent translation of the RNA. This invention alsoprovides an antisense oligonucleotide having a sequence capable ofspecifically hybridizing to genomic DNA encoding a mammalian NPFFreceptor, so as to prevent translation of the genomic DNA. In oneembodiment, the oligonucleotide comprises chemically modifiednucleotides or nucleotide analogues.

This invention provides an antibody capable of binding to a mammalianNPFF receptor encoded by a nucleic acid encoding a mammalian NPFFreceptor. In one embodiment, the mammalian NPFF receptor is a rat NPFF1receptor. In another embodiment, the mammalian NPFF receptor is a humanNPFF1 receptor. In a further embodiment, the mammalian NPFF receptor isa human NPFF2 receptor. In a further embodiment, the mammalian NPFFreceptor is a rat NPFF2 receptor. This invention also provides an agentcapable of competitively inhibiting the binding of the antibody to amammalian NPFF receptor. In one embodiment, the antibody is a monoclonalantibody or antisera.

This invention provides a pharmaceutical composition comprising (a) anamount of the oligonucleotide capable of passing through a cell membraneand effective to reduce expression of a mammalian NPFF receptor and (b)a pharmaceutically acceptable carrier capable of passing through thecell membrane. In an embodiment, the oligonucleotide is coupled to asubstance which inactivates mRNA. In a further embodiment, the substancewhich inactivates mRNA is a ribozyme. In another embodiment, thepharmaceutically acceptable carrier comprises a structure which binds toa mammalian NPFF receptor on a cell capable of being taken up by thecells after binding to the structure. In a further embodiment, thepharmaceutically acceptable carrier is capable of binding to a mammalianNPFF receptor which is specific for a selected cell type.

This invention provides a pharmaceutical composition which comprises anamount of an antibody effective to block binding of a ligand to a humanNPFF receptor and a pharmaceutically acceptable carrier.

As used herein, the phrase “pharmaceutically acceptable carrier” meansany of the standard pharmaceutically acceptable carriers. Examplesinclude, but are not limited to, phosphate buffered saline,physiological saline, water, and emulsions, such as oil/water emulsions.

This invention provides a transgenic, nonhuman mammal expressing DNAencoding a mammalian NPFF receptor. This invention also provides atransgenic, nonhuman mammal comprising a homologous recombinationknockout of the native mammalian NPFF receptor. This invention furtherprovides a transgenic, nonhuman mammal whose genome comprises antisenseDNA complementary to the DNA encoding a mammalian NPFF receptor soplaced within the genome as to be transcribed into antisense mRNA whichis complementary to mRNA encoding the mammalian NPFF receptor and whichhybridizes to mRNA encoding the mammalian NPFF receptor, therebyreducing its translation. In an embodiment, the DNA encoding themammalian NPFF receptor additionally comprises an inducible promoter. Inanother embodiment, the DNA encoding the mammalian NPFF receptoradditionally comprises tissue specific regulatory elements. In a furtherembodiment, the transgenic, nonhuman mammal is a mouse.

Animal model systems which elucidate the physiological and behavioralroles of the polypeptides of this invention are produced by creatingtransgenic animals in which the activity of the polypeptide is eitherincreased or decreased, or the amino acid sequence of the expressedpolypeptide is altered, by a variety of techniques. Examples of thesetechniques include, but are not limited to: 1) Insertion of normal ormutant versions of DNA encoding the polypeptide, by microinjection,electroporation, retroviral transfection or other means well known tothose in the art, into appropriate fertilized embryos in order toproduce a transgenic animal or 2) Homologous recombination of mutant ornormal, human or animal versions of these genes with the native genelocus in transgenic animals to alter the regulation of expression or thestructure of these polypeptide sequences. The technique of homologousrecombination is well known in the art. It replaces the native gene withthe inserted gene and so is useful for producing an animal that cannotexpress native polypeptides but does express, for example, an insertedmutant polypeptide, which has replaced the native polypeptide in theanimal's genome by recombination, resulting in underexpression of thetransporter. Microinjection adds genes to the genome, but does notremove them, and so is useful for producing an animal which expressesits own and added polypeptides, resulting in overexpression of thepolypeptides.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium. DNA or cDNA encoding apolypeptide of this invention is purified from a vector by methods wellknown in the art. Inducible promoters may be fused with the codingregion of the DNA to provide an experimental means to regulateexpression of the trans-gene. Alternatively, or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipet puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only for exemplary purposes.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian NPFF receptor which comprisescontacting cells containing DNA encoding and expressing on their cellsurface the mammalian NPFF receptor, wherein such cells do not normallyexpress the mammalian NPFF receptor, with the compound under conditionssuitable for binding, and detecting specific binding of the chemicalcompound to the mammalian NPFF receptor. This invention also provides aprocess for identifying a chemical compound which specifically binds toa mammalian NPFF receptor which comprises contacting a membrane fractionfrom a cell extract of cells containing DNA encoding and expressing ontheir cell surface the mammalian NPFF receptor, wherein such cells donot normally express the mammalian NPFF receptor, with the compoundunder conditions suitable for binding, and detecting specific binding ofthe chemical compound to the mammalian NPFF receptor. In one embodiment,the NPFF receptor is a NPFF1 receptor. In a further embodiment, themammalian NPFF1 receptor is a rat NPFF1 receptor. In another embodiment,the mammalian NPFF1 receptor is a human NPFF1 receptor. In oneembodiment, the mammalian NPFF receptor is a NPFF2 receptor. In afurther embodiment, the mammalian NPFF2 receptor is a human NPFF2receptor. In a further embodiment, the mammalian NPFF2 receptor is a ratNPFF2 receptor. In another embodiment, the mammalian NPFF receptor hassubstantially the same amino acid sequence as the NPFF receptor encodedby plasmid pEXJ-rNPFF1, plasmid pWE15-hNPFF1, plasmid pcDNA3.1-hNPFF2b,plasmid pcDNA3.1-hNPFF1, or plasmid pcDNA3.1-rNPFF2-f. In a furtherembodiment, the mammalian NPFF receptor has substantially the same aminoacid sequence as that shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8 or SEQ ID NO:44. In another embodiment, the mammalian NPFFreceptor has the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:44. In one embodiment, thecompound is not previously known to bind to a mammalian NPFF receptor.This invention further provides a compound identified by theabove-described processes.

In one embodiment of the above-described processes, the cell is aninsect cell. In another embodiment, the cell is a mammalian cell. In afurther embodiment, the cell is normeuronal in origin. In a furtherembodiment, the normeuronal cell is a COS-7 cell, 293 human embryonickidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−)cell. In an embodiment, the compound is a compound not previously knownto bind to a mammalian NPFF receptor. This invention also provides acompound identified by the above-described process.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianNPFF receptor which comprises separately contacting cells expressing ontheir cell surface the mammalian NPFF receptor, wherein such cells donot normally express the mammalian NPFF receptor, with both the chemicalcompound and a second chemical compound known to bind to the receptor,and with only the second chemical compound, under conditions suitablefor binding of both compounds, and detecting specific binding of thechemical compound to the mammalian NPFF receptor, a decrease in thebinding of the second chemical compound to the mammalian NPFF receptorin the presence of the chemical compound indicating that the chemicalcompound binds to the mammalian NPFF receptor.

This invention also provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianNPFF receptor which comprises separately contacting a membranepreparation from cells expressing on their cell surface the mammalianNPFF receptor, wherein such cells do not normally express the mammalianNPFF receptor, with both the chemical compound and a second chemicalcompound known to bind to the receptor, and with only the secondchemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian NPFF receptor, a decrease in the binding of the secondchemical compound to the mammalian NPFF receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian NPFF receptor.

In an embodiment of the present invention, the second chemical compoundis NPFF or a homolog or analog of NPFF.

In one embodiment, the mammalian NPFF receptor is a NPFF1 receptor. In afurther embodiment, the mammalian NPFF1 receptor is a rat NPFF1receptor. In another embodiment, the mammalian NPFF1 receptor is a humanNPFF1 receptor. In another embodiment, the mammalian NPFF receptor is aNPFF2 receptor. In a further embodiment, the NPFF2 receptor is a humanNPFF2 receptor. In a further embodiment, the NPFF2 receptor is a ratNPFF2 receptor. In another embodiment, the mammalian NPFF receptor hassubstantially the same amino acid sequence as the NPFF receptor encodedby plasmid pEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1or pcDNA3.1-rNPFF2-f. In a further embodiment, the mammalian NPFFreceptor has substantially the same amino acid sequence as that shown inSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:44. Inanother embodiment, the mammalian NPFF receptor has the amino acidsequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 orSEQ ID NO:44.

In one embodiment, the cell is an insect cell. In another embodiment,the cell is a mammalian cell. In a further embodiment, the cell isnormeuronal in origin. In another embodiment, the normeuronal cell is aCOS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell,a mouse Y1 cell, or a LM(tk−) cell. In one embodiment, the compound isnot previously known to bind to a mammalian NPFF receptor.

This invention provides a compound identified by the above-describedprocesses.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian NPFF receptor to identify acompound which specifically binds to the mammalian NPFF receptor, whichcomprises (a) contacting cells transfected with and expressing DNAencoding the mammalian NPFF receptor with a compound known to bindspecifically to the mammalian NPFF receptor; (b) contacting thepreparation of step (a) with the plurality of compounds not known tobind specifically to the mammalian NPFF receptor, under conditionspermitting binding of compounds known to bind the mammalian NPFFreceptor; (c) determining whether the binding of the compound known tobind to the mammalian NPFF receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian NPFF receptor to identify acompound which specifically binds to the mammalian NPFF receptor, whichcomprises (a) contacting a membrane preparation from cells transfectedwith and expressing DNA encoding the mammalian NPFF receptor with theplurality of compounds not known to bind specifically to the mammalianNPFF receptor under conditions permitting binding of compounds known tobind to the mammalian NPFF receptor; (b) determining whether the bindingof a compound known to bind to the mammalian NPFF receptor is reduced inthe presence of any compound within the plurality of compounds, relativeto the binding of the compound in the absence of the plurality ofcompounds; and if so (c) separately determining the binding to themammalian NPFF receptor of compounds included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the mammalian NPFF receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian NPFF receptor to identify acompound which specifically binds to the mammalian NPFF receptor, whichcomprises (a) contacting a membrane preparation from cells transfectedwith and expressing the mammalian NPFF receptor with a compound known tobind specifically to the mammalian NPFF receptor; (b) contacting thepreparation of step (a) with the plurality of compounds not known tobind specifically to the mammalian NPFF receptor, under conditionspermitting binding of compounds known to bind the mammalian NPFFreceptor; (c) determining whether the binding of the compound known tobind to the mammalian NPFF receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian NPFF receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian NPFFreceptor.

In one embodiment of the above-described methods, the mammalian NPFFreceptor is a NPFF1 receptor. In a further embodiment, the mammalianNPFF1 receptor is a rat NPFF1 receptor. In another embodiment, themammalian NPFF1 receptor is a human NPFF1 receptor. In anotherembodiment, the mammalian NPFF receptor is a NPFF2 receptor. In afurther embodiment the NPFF2 receptor is a human NPFF2 receptor. In afurther embodiment, the mammalian NPFF2 receptor is a rat NPFF2receptor. In another embodiment, the cell is a mammalian cell. In afurther embodiment, the mammalian cell is non-neuronal in origin. Inanother embodiment, the non-neuronal cell is a COS-7 cell, a 293 humanembryonic kidney cell, a LM(tk−) cell, a CHO cell, a mouse Y1 cell, oran NIH-3T3 cell.

This invention also provides a method of detecting expression of amammalian NPFF receptor by detecting the presence of mRNA coding for themammalian NPFF receptor which comprises obtaining total mRNA from thecell and contacting the mRNA so obtained from a nucleic acid probe underhybridizing conditions, detecting the presence of mRNA hybridizing tothe probe, and thereby detecting the expression of the mammalian NPFFreceptor by the cell.

This invention further provides a method of detecting the presence of amammalian NPFF receptor on the surface of a cell which comprisescontacting the cell with an antibody under conditions permitting bindingof the antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of the mammalianNPFF receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian NPFF receptors whichcomprises producing a transgenic, nonhuman mammal whose levels ofmammalian NPFF receptor activity are varied by use of an induciblepromoter which regulates mammalian NPFF receptor expression.

This invention also provides a method of determining the physiologicaleffects of varying levels of activity of mammalian NPFF receptors whichcomprises producing a panel of transgenic, nonhuman mammals eachexpressing a different amount of mammalian NPFF receptor.

This invention provides a method for identifying an antagonist capableof alleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a mammalian NPFF receptor comprisingadministering a compound to a transgenic, nonhuman mammal, anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic, nonhuman mammal as a resultof overactivity of a mammalian NPFF receptor, the alleviation of theabnormality identifying the compound as an antagonist. This inventionalso provides an antagonist identified by the above-described method.This invention further provides a pharmaceutical composition comprisingan antagonist identified by the above-described method and apharmaceutically acceptable carrier. This invention provides a method oftreating an abnormality in a subject wherein the abnormality isalleviated by decreasing the activity of a mammalian NPFF receptor whichcomprises administering to the subject an effective amount of thispharmaceutical composition, thereby treating the abnormality.

This invention provides a method for identifying an agonist capable ofalleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a mammalian NPFF receptorcomprising administering a compound to transgenic, nonhuman mammal, anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic, nonhuman mammal, thealleviation of the abnormality identifying the compound as an agonist.This invention also provides an agonist identified by theabove-described method. This invention further provides a pharmaceuticalcomposition comprising an agonist identified by the above-describedmethod and a pharmaceutically acceptable carrier. This invention furtherprovides a method of treating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a mammalian NPFFreceptor which comprises administering to the subject an effectiveamount of this pharmaceutical composition, thereby treating theabnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific mammalian allelewhich comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a mammalian NPFF receptor and labeled with a detectable marker;(e) detecting labeled bands which have hybridized to the DNA encoding amammalian NPFF receptor labeled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and(g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step (e) and the DNA obtained fordiagnosis from step (f) to determine whether the patterns are the sameor different and to diagnose thereby predisposition to the disorder ifthe patterns are the same. In one embodiment, a disorder associated withthe activity of a specific mammalian allele is diagnosed.

This invention provides a method of preparing the purified mammalianNPFF receptor which comprises: (a) inducing cells to express themammalian NPFF receptor; (b) recovering the mammalian NPFF receptor fromthe induced cells; and (c) purifying the mammalian NPFF receptor sorecovered.

This invention provides a method of preparing the purified mammalianNPFF receptor which comprises: (a) inserting nucleic acid encoding themammalian NPFF receptor in a suitable vector; (b) introducing theresulting vector in a suitable host cell; (c) placing the resulting cellin suitable condition permitting the production of the isolatedmammalian NPFF receptor; (d) recovering the mammalian NPFF receptorproduced by the resulting cell; and (e) purifying the mammalian NPFFreceptor so recovered.

This invention provides a process for determining whether a chemicalcompound is a mammalian NPFF receptor agonist which comprises contactingcells transfected with and expressing DNA encoding the mammalian NPFFreceptor with the compound under conditions permitting the activation ofthe mammalian NPFF receptor, and detecting an increase in mammalian NPFFreceptor activity, so as to thereby determine whether the compound is amammalian NPFF receptor agonist. This invention also provides a processfor determining whether a chemical compound is a mammalian NPFF1receptor antagonist which comprises contacting cells transfected withand expressing DNA encoding the mammalian NPFF receptor with thecompound in the presence of a known mammalian NPFF receptor agonist,under conditions permitting the activation of the mammalian NPFFreceptor, and detecting a decrease in mammalian NPFF receptor activity,so as to thereby determine whether the compound is a mammalian NPFFreceptor antagonist. In one embodiment, the mammalian NPFF receptor is aNPFF1 receptor. In a further embodiment, the mammalian NPFF1 receptor isa rat NPFF1 receptor. In another embodiment, the mammalian NPFF1receptor is a human NPFF1 receptor. In one embodiment, the mammalianNPFF receptor is a NPFF2 receptor. In a further embodiment, themammalian NPFF2 receptor is a human NPFF2 receptor. In a furtherembodiment, the mammalian NPFF2 receptor is a rat NPFF2 receptor.

This invention further provides a pharmaceutical composition whichcomprises an amount of a mammalian NPFF receptor agonist determined bythe above-described process effective to increase activity of amammalian NPFF receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian NPFF receptor agonist is not previouslyknown.

This invention provides a pharmaceutical composition which comprises anamount of a mammalian NPFF receptor antagonist determined by theabove-described process effective to reduce activity of a mammalian NPFFreceptor and a pharmaceutically acceptable carrier. In one embodiment,the mammalian NPFF receptor antagonist is not previously known.

This invention provides a process for determining whether a chemicalcompound specifically binds to and activates a mammalian NPFF receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the mammalian NPFF receptor,wherein such cells do not normally express the mammalian NPFF receptor,with the chemical compound under conditions suitable for activation ofthe mammalian NPFF receptor, and measuring the second messenger responsein the presence and in the absence of the chemical compound, a change inthe second messenger response in the presence of the chemical compoundindicating that the compound activates the mammalian NPFF receptor. Inone embodiment, the second messenger response comprises chloride channelactivation and the change in second messenger is an increase in thelevel of inward chloride current.

This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of amammalian NPFF receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian NPFF receptor, wherein such cells do not normallyexpress the mammalian NPFF receptor, with both the chemical compound anda second chemical compound known to activate the mammalian NPFFreceptor, and with only the second chemical compound, under conditionssuitable for activation of the mammalian NPFF receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian NPFF receptor. In one embodiment, the secondmessenger response comprises chloride channel activation and the changein second messenger response is a smaller increase in the level ofinward chloride current in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound. This invention also provides the above-describedprocesses performed with membrane preparations from cells producing asecond messenger response and transfected with and expressing themammalian NPFF receptor.

In one embodiment of the above-described processes, the mammalian NPFFreceptor is a NPFF1 receptor. In a further embodiment, the mammalianNPFF1 receptor is a rat NPFF1 receptor. In another embodiment, themammalian NPFF1 receptor is a human NPFF1 receptor. In anotherembodiment, the mammalian NPFF receptor is a NPFF2 receptor. In afurther embodiment, the mammalian NPFF2 receptor is a human NPFF2receptor. In a further embodiment, the mammalian NPFF2 receptor is a ratNPFF2 receptor. In another embodiment, the mammalian NPFF receptor hassubstantially the same amino acid sequence as encoded by the plasmidpEXJ-rNPFF1, pWE15-hNPFF1, pcDNA3.1-hNPFF2b, pcDNA3.1-hNPFF1 orpcDNA3.1-rNPFF2-f. In a further embodiment, the mammalian NPFF receptorhas substantially the same amino acid sequence as that shown in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:44. In anotherembodiment, the mammalian NPFF receptor has an amino acid sequenceidentical to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:44. In an embodiment, the cell isan insect cell. In a further embodiment, the cell is a mammalian cell.In a still further embodiment, the mammalian cell is normeuronal inorigin. In another embodiment, the normeuronal cell is a COS-7 cell, CHOcell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk−) cell. Inan embodiment, the compound is not previously known to bind to amammalian NPFF receptor. This invention also provides a compounddetermined by the above-described processes.

This invention also provides a pharmaceutical composition whichcomprises an amount of a mammalian NPFF receptor agonist determined bythe above-described processes effective to increase activity of amammalian NPFF receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian NPFF receptor agonist is not previouslyknown.

This invention further provides a pharmaceutical composition whichcomprises an amount of a mammalian NPFF receptor antagonist determinedby the above-described processes effective to reduce activity of amammalian NPFF receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian NPFF receptor antagonist is not previouslyknown.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a mammalian NPFF receptor to identify acompound which activates the mammalian NPFF receptor which comprises:

(a) contacting cells transfected with and expressing the mammalian NPFFreceptor with the plurality of compounds not known to activate themammalian NPFF receptor, under conditions permitting activation of themammalian NPFF receptor; (b) determining whether the activity of themammalian NPFF receptor is increased in the presence of the compounds;and if so (c) separately determining whether the activation of themammalian NPFF receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the mammalian NPFF receptor. In one embodiment, the mammalianNPFF receptor is a human NPFF receptor. In a further embodiment thehuman NPFF receptor is a human NPFF1 receptor or a human NPFF2 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian NPFFreceptor to identify a compound which inhibits the activation of themammalian NPFF receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian NPFF receptor with theplurality of compounds in the presence of a known mammalian NPFFreceptor agonist, under conditions permitting activation of themammalian NPFF receptor; (b) determining whether the activation of themammalian NPFF receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian NPFF receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian NPFF receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian NPFF receptor. In one embodiment, the mammalian NPFF receptoris a NPFF1 receptor. In a further embodiment, the mammalian NPFF1receptor is a rat NPFF1 receptor. In another embodiment, the NPFF1receptor is a human NPFF1 receptor. In another embodiment, the mammalianNPFF receptor is a NPFF2 receptor. In a further embodiment, the NPFF2receptor is a human NPFF2 receptor. In a further embodiment, themammalian NPFF2 receptor is a rat NPFF2 receptor.

In one embodiment of the above-described methods, the cell is amammalian cell. In another embodiment, the mammalian cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell or anNIH-3T3 cell.

This invention provides a pharmaceutical composition comprising acompound identified by the above-described methods effective to increasemammalian NPFF receptor activity and a pharmaceutically acceptablecarrier.

This invention also provides a pharmaceutical composition comprising acompound identified by the above-described methods effective to decreasemammalian NPFF receptor activity and a pharmaceutically acceptablecarrier.

This invention further provides a method of measuring polypeptideactivation in an oocyte expression system such as a Xenopus oocyteexpression system or melanophore. In an embodiment, polypeptideactivation is determined by measurement of ion channel activity. Inanother embodiment, polypeptide activation is measured by aequerinluminescence.

Expression of genes in Xenopus oocytes is well known in the art(Coleman, A., 1984; Masu, Y., et al., 1994) and is performed usingmicroinjection of native mRNA or in vitro synthesized mRNA into frogoocytes. The preparation of in vitro synthesized mRNA can be performedby various standard techniques (Sambrook, et al. 1989) including usingT7 polymerase with the mCAP RNA mapping kit (Stratagene).

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated diseases. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data (McKusick, V.A., 1998). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes). Thedifference in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian NPFF receptor which comprises administering to the subject anamount of a compound which is a mammalian NPFF receptor agonisteffective to treat the abnormality. In separate embodiments, theabnormality is a lower urinary tract disorder such as interstitialcystitis or urinary incontinence such as urge incontinence or stressincontinence particularly stress incontinence, a regulation of a steroidhormone disorder, an epinephrine release disorder, a gastrointestinaldisorder, irritable bowel syndrome, a cardiovascular disorder, anelectrolyte balance disorder, diuresis, hypertension, hypotension,diabetes, hypoglycemia, a respiratory disorder, asthma, a reproductivefunction disorder, an immune disorder, an endocrine disorder, amusculoskeletal disorder, a neuroendocrine disorder, a cognitivedisorder, a memory disorder, a sensory modulation and transmissiondisorder, a motor coordination disorder, a sensory integration disorder,a motor integration disorder, a dopaminergic function disorder, aserotonergic function disorder, an appetite disorder, obesity, a sensorytransmission disorder, an olfaction disorder, a sympathetic innervationdisorder, an affective disorder, pain, psychotic behavior, morphinetolerance, nicotine addiction, opiate addiction, or migraine.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of amammalian NPFF receptor which comprises administering to the subject anamount of a compound which is a mammalian NPFF receptor antagonisteffective to treat the abnormality. In separate embodiments, theabnormality is a lower urinary tract disorder such as interstitialcystitis or urinary incontinence such as urge incontinence or stressincontinence particularly stress incontinence, a regulation of a steroidhormone disorder, an epinephrine release disorder, a gastrointestinaldisorder, irritable bowel syndrome, a cardiovascular disorder, anelectrolyte balance disorder, diuresis, hypertension, hypotension,diabetes, hypoglycemia, a respiratory disorder, asthma, a reproductivefunction disorder, an immune disorder, an endocrine disorder, amusculoskeletal disorder, a neuroendocrine disorder, a cognitivedisorder, a memory disorder, a sensory modulation and transmissiondisorder, a motor coordination disorder, a sensory integration disorder,a motor integration disorder, a dopaminergic function disorder, aserotonergic function disorder, an appetite disorder, obesity, a sensorytransmission disorder, an olfaction disorder, a sympathetic innervationdisorder, an affective disorder, pain, psychotic behavior, morphinetolerance, nicotine addiction, opiate addiction, or migraine.

This invention provides a method of treating urinary incontinence whichcomprises administering to a subject an amount of an antagonist of ahuman NPFF2 receptor effective to inhibit activation of the receptor andthereby treat incontinence, such as urge incontinence or stressincontinence.

This invention provides a method of treating urinary retention whichcomprises administering to a subject an amount of an agonist of a humanNPFF2 receptor effective to activate the receptor and thereby treatretention.

This invention provides a method of treating hypertension whichcomprises administering to a subject an amount of an antagonist of ahuman NPFF1 receptor effective to inhibit activation of the receptor andthereby treat hypertension.

This invention provides a method of treating hypotension which comprisesadministering to a subject an amount of an agonist of a human NPFF1receptor effective to activate the receptor and thereby treathypotension.

This invention provides a method of modifying the feeding behavior of asubject which comprises administering the subject an amount of acompound which is a NPFF2 receptor agonist effective to decrease theconsumption of food by the subject so as to thereby modify the feedingbehavior of the subject.

In one embodiment of the above-described method the subject is avertebrate. In another embodiment, the subject is a mammal. In a furtherembodiment, the subject is a human. In another embodiment, the subjectis a canine.

This invention also provides a method of treating a feeding disorder ina subject which comprises administering to the subject an amount of acompound which is NPFF2 receptor agonist effective to activate thereceptor and thereby treat the feeding disorder. In separateembodiments, the feeding disorder is bulimia, bulimia nervosa orobesity.

In one embodiment of the above-described method the subject is avertebrate. In another embodiment, the subject is a mammal. In a furtherembodiment, the subject is a human. In another embodiment, the subjectis a canine.

This invention further provides a method of inhibiting feeding whichcomprises administering to a subject an amount of an agonist of a humanNPFF2 receptor effective to activate the receptor and thereby inhibitfeeding.

This invention also provides the use of mammalian NPFF receptors foranalgesia.

This invention provides a process for making a composition of matterwhich specifically binds to a mammalian NPFF receptor which comprisesidentifying a chemical compound using any of the processes describedherein for identifying a compound which binds to and/or activates orinhibits activation of a mammalian NPFF receptor and then synthesizingthe chemical compound or a novel structural and functional analog orhomolog thereof. In one embodiment, the mammalian NPFF receptor is ahuman NPFF1 receptor. In another embodiment, the mammalian NPFF receptoris a human NPFF2 receptor.

This invention further provides a process for preparing a pharmaceuticalcomposition which comprises admixing a pharmaceutically acceptablecarrier and a pharmaceutically acceptable amount of a chemical compoundidentified by any of the processes described herein for identifying acompound which binds to and/or activates or inhibits activation of amammalian NPFF receptor or a novel structural and functional analog orhomolog thereof. In one embodiment, the mammalian NPFF receptor is ahuman NPFF1 receptor. In another embodiment, the mammalian NPFF receptoris a human NPFF2 receptor.

This invention provides for use of a human NPFF2 receptor antagonist forthe preparation of a pharmaceutical composition for treating urinaryincontinence, such as urge incontinence or stress incontinence. Thisinvention provides for use of a human NPFF2 receptor agonist for thepreparation of a pharmaceutical composition for treating urinaryretention.

This invention provides for use of a human NPFF1 receptor antagonist forthe preparation of a pharmaceutical composition for treatinghypertension. This invention provides for use of a human NPFF1 receptoragonist for the preparation of a pharmaceutical composition for treatinghypotension.

This invention also provides the use of a human NPFF2 receptor agonistfor the preparation of a pharmaceutical composition for inhibitingfeeding. This invention provides the use of a human NPFF2 receptoragonist for the preparation of a pharmaceutical composition for treatinga feeding disorder.

Thus, once the gene for a targeted receptor subtype is cloned, it isplaced into a recipient cell which then expresses the targeted receptorsubtype on its surface. This cell, which expresses a single populationof the targeted human receptor subtype, is then propagated resulting inthe establishment of a cell line. This cell line, which constitutes adrug discovery system, is used in two different types of assays: bindingassays and functional assays. In binding assays, the affinity of acompound for both the receptor subtype that is the target of aparticular drug discovery program and other receptor subtypes that couldbe associated with side effects are measured. These measurements enableone to predict the potency of a compound, as well as the degree ofselectivity that the compound has for the targeted receptor subtype overother receptor subtypes. The data obtained from binding assays alsoenable chemists to design compounds toward or away from one or more ofthe relevant subtypes, as appropriate, for optimal therapeutic efficacy.In functional assays, the nature of the response of the receptor subtypeto the compound is determined. Data from the functional assays showwhether the compound is acting to inhibit or enhance the activity of thereceptor subtype, thus enabling pharmacologists to evaluate compoundsrapidly at their ultimate human receptor subtypes targets permittingchemists to rationally design drugs that will be more effective and havefewer or substantially less severe side effects than existing drugs.

Approaches to designing and synthesizing receptor subtype-selectivecompounds are well known and include traditional medicinal chemistry andthe newer technology of combinatorial chemistry, both of which aresupported by computer-assisted molecular modeling. With such approaches,chemists and pharmacologists use their knowledge of the structures ofthe targeted receptor subtype and compounds determined to bind and/oractivate or inhibit activation of the receptor subtype to design andsynthesize structures that will have activity at these receptorsubtypes.

Combinatorial chemistry involves automated synthesis of a variety ofnovel compounds by assembling them using different combinations ofchemical building blocks. The use of combinatorial chemistry greatlyaccelerates the process of generating compounds. The resulting arrays ofcompounds are called libraries and are used to screen for compounds(“lead compounds”) that demonstrate a sufficient level of activity atreceptors of interest. Using combinatorial chemistry it is possible tosynthesize “focused” libraries of compounds anticipated to be highlybiased toward the receptor target of interest.

Once lead compounds are identified, whether through the use ofcombinatorial chemistry or traditional medicinal chemistry or otherwise,a variety of homologs and analogs are prepared to facilitate anunderstanding of the relationship between chemical structure andbiological or functional activity. These studies define structureactivity relationships which are then used to design drugs with improvedpotency, selectivity and pharmacokinetic properties. Combinatorialchemistry is also used to rapidly generate a variety of structures forlead optimization. Traditional medicinal chemistry, which involves thesynthesis of compounds one at a time, is also used for furtherrefinement and to generate compounds not accessible by automatedtechniques. Once such drugs are defined the production is scaled upusing standard chemical manufacturing methodologies utilized throughoutthe pharmaceutical and chemistry industry.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS

Materials and Methods

Cloning of rat and human NPFF1 receptor MOPAC (Mixed OligonucleotidePrimed Amplification of cDNA

100 ng of rat genomic DNA (Clonetech, Palo Alto, Calif.) was used fordegenerate MOPAC PCR using Taq DNA polymerase (Boehringer-Mannheim,Indianapolis, Ind.) and the following degenerate oligonucleotides:JAB126, designed based on an alignment of the sixth transmembrane domainof more than 180 members of the rhodopsin superfamily of Gprotein-coupled receptors; and JAB108, designed based on an alignment ofthe seventh transmembrane domain of the same rhodopsin superfamily.

The conditions for the MOPAC PCR reaction were as follows: 3 minute holdat 94° C.; 10 cycles of 1 minute at 94° C., 1 minute 45 seconds at 44°C., 2 minutes at 72° C.; 30 cycles of 94° C. for 1 minute, 49° C. for 1minute 45 seconds, 2 minutes at 72° C.; 4 minute hold at 72° C.; 4° C.until ready for agarose gel electrophoresis.

The products were run on a 1% agarose TAE gel and bands of the expectedsize (^(˜)150 bp) were cut from the gel, purified using the QIAQUICK gelextraction kit (QIAGEN, Chatsworth, Calif.), and subcloned into the TAcloning vector (Invitrogen, San Diego, Calif.). White(insert-containing) colonies were picked and subjected to PCR usingpCR2.1 vector primers JAB1 and JAB2 using the Expand Long Template PCRSystem and the following protocol: 94° C. hold for 3 minutes; 35 cyclesof 94° C. for 1 minute, 68° C. for 1 minute 15 seconds; 2 minute hold at68° C., 4° C. hold until products were ready for purification. PCRproducts were purified by isopropanol precipitation (10 μl PCR product,18 μl low TE, 10.5 μl 2M NaClO₄ and 21.5 μl isopropanol) and sequencedusing the ABI Big Dye cycle sequencing protocol and ABI 377 sequencers(ABI, Foster City, Calif.). Nucleotide and amino acid sequence analyseswere performed using the Wisconsin Package (GCG, Genetics ComputerGroup, Madison, Wis.). Two PCR products produced from rat genomic cDNA(MPR3-RGEN-31 and MPR3—RGEN-45) were determined to be identical clonesof a novel G protein-coupled receptor-like sequence based on databasesearches and its homology to other known G protein-coupled receptors(^(˜)30-40% amino acid identity to dopamine D2, orexin, galanin,angiotensin 1 and 5-HT_(2b) receptors). This novel sequence wasdesignated SNORF2.

Cloning of the Full-Length Coding Sequence of SNORF2 (Rat NPFF1)

Pools of the rat hypothalamic cDNA library “I” were screened by PCR withSNORF2-specific primers JAB208 and JAB209 and the Expand Long TemplatePCR system (Boehringer-Mannheim, Indianapolis, Ind.) with the followingPCR protocol: 94° C. hold for 3 minutes; 40 cycles of 94° C. for 1minute, 68° C. for 2 minutes; 4 minute hold at 68° C.; 4° C. hold untilthe samples are run on a gel. This screen yielded a positive pool I36Eand a positive sub-pool I36E-17. High stringency hybridization ofisolated colonies from I36E-17 with the SNORF2-specific oligonucleotideprobe JAB211 and subsequent PCR testing of positive colonies indicatedthat the isolated clone I36E-17-1B-1 contained at least a partial cloneof SNORF2. Sequencing of I36E-17-1B-1 revealed that this insertcontained the coding region from the TMIII-TMIV loop through the stopcodon, including some 3′ untranslated sequence. From this sequence, anew forward primer, JAB221, was designed in TMV. PCR screening of asecond rat hypothalamic cDNA library “J” with primers JAB221 and JAB209,and subsequent colony hybridization with the JAB211 probe on a lowcomplexity positive sub-pool resulted in the isolation of a SNORF2 cloneJ-13-16-A1. Full-length double-stranded sequence of SNORF2 wasdetermined by sequencing both strands of the J-13-16-A1 plasmid using anABI 377 sequencer as described above. This insert is about 2.8 kb inlength with an approximately 200 bp 5′ untranslated region, a 1296 bpcoding region, and a 1.3 kb 3'untranslated region. The clone is also inthe correct orientation for expression in the mammalian expressionvector pEXJ.T7. This construct of SNORF2 in pEXJ.T7 was designated BN-6.The full length SNORF2 was determined to be most like the orexin 1receptor (45% DNA identity, 35% amino acid identity), orexin 2 receptor(40% DNA identity, 32% amino acid identity), and NPY2 receptor (47% DNAidentity, 29% amino acid identity), although several other Gprotein-coupled receptors also displayed significant homology. Therewere no sequences in the Genbank databases (genembl, sts, est, gss, orswissprot) that were identical to SNORF2. SNORF2 also showed significanthomology (85% nucleotide identity, 93% amino acid identity) to a partialG protein-coupled receptor fragment in the Synaptic PharmaceuticalCorporation in-house database, designated PLC29b. PLC29b, which includespart of the amino terminus through TMIII, was originally isolated from ahuman genomic library using oligonucleotide probes for NPY4. Subsequentscreening of a human hippocampal cDNA library yielded an overlappingsequence extending into TMIV. Based on sequence similarity, this humansequence appears to be a partial clone of the human homolog of SNORF2.

The following is a list of primers and their associated sequences whichwere used in the cloning of these receptors:

(SEQ ID NO: 9) JAB126: 5′-GYNTWYRYNNTNWSNTGGHTNCC-3′ (SEQ ID NO: 10)JAB108: 5′-AVNADNGBRWAVANNANNGGRTT-3′ (SEQ ID NO: 11) JAB1:5′-TTATGCTTCCGGCTCGTATGTTGTG-3′ (SEQ ID NO: 12) JAB2:5′-ATGTGCTGCAAGGCGATTAAGTTGGG-3′ (SEQ ID NO: 13) JAB208:5′-GGTGCTGCTGCTGCTCATCGACTATG-3′ (SEQ ID NO: 14) JAB209:5′-TTGGCGCTGCTGTGGAAGAAGGCCAG-3′ (SEQ ID NO: 15) JAB221:5′-CGGTGCTCTTCGCGCACATCTACC-3′ (SEQ ID NO: 16) JAB211:5′-TGCCAAGGGGAAGGCGTAGACCGACAGCAGGTGCAGTTG CAGCTCGATCAGCTCCCCATA-3′Isolation of the Full-Length Human SNORF2 Receptor Gene (Human NPFF1)

The full-length, intronless version of the human NPFF1 receptor gene maybe isolated using standard molecular biology techniques and approachessuch as those briefly described below:

Approach #1: To obtain a full-length human NPFF1 receptor, a humancosmid library was screened with a ³²P-labeled oligonucleotide probe,BB609, corresponding to the ⅔ loop of the PLC29b clone. A positive clonewas isolated and partially sequenced, revealing part of the aminoterminus and TMs I and II.

The full-length sequence may be obtained by sequencing this cosmid clonewith additional sequencing primers. Since at least two introns arepresent in this gene, one in the amino terminus and one just after thethird transmembrane domain, the full-length intronless gene may beobtained from cDNA using standard molecular biology techniques. Forexample, a forward PCR primer designed in the 5′UT and a reverse PCRprimer designed in the 3′UT may be used to amplify a full-length,intronless gene from cDNA. RT-PCR localization has identified severalhuman tissues which could be used for this purpose, includingcerebellum, spinal cord, hippocampus, lung and kidney. Standardmolecular biology techniques could be used to subclone this gene into amammalian expression vector.

Approach #2: Standard molecular biology techniques could be used toscreen commercial human cDNA phage libraries by hybridization under highstringency with a ³²P-labeled oligonucleotide probe, BB609,corresponding to the ⅔ loop of the PLC29b clone. One may isolate afull-length human NPFF1 by obtaining a plaque purified clone from thelambda libraries and then subjecting the clone to direct DNA sequencingusing primers from the PLC29b sequence. Alternatively, standardmolecular biology techniques could be used to screen in-house human cDNAplasmid libraries by PCR amplification of library pools using primers tothe human NPFF1 sequence (BB629, forward primer in TMI, and A71, reverseprimer in TMIV). A full-length clone could be isolated by Southernhybridization of colony lifts of positive pools with a ³²P-labeledoligonucleotide probe, BB609, corresponding to the ⅔ loop of the PLC29bclone.

Approach #3: As yet another alternative method, one could utilize 3′ and5′ RACE to generate PCR products from human cDNA expressing human NPFF1(for example, cerebellum, spinal cord, hippocampus, lung and kidney),which contain the additional sequences of human NPFF1. For 5′ RACE, areverse primer derived from PLC29b between the amino terminus and TM IVcould be used to amplify the additional amino terminus sequence forhNPFF1. For 3′ RACE, a forward primer derived from PLC29b between theamino terminus and TM IV could be used to amplify the additional 3′sequence for hNPFF1, including TMs 5-7 and the COOH terminus. These RACEPCR product could then be sequenced to determine the missing sequence.This new sequence could then be used to design a forward PCR primer inthe 5′UT and a reverse primer in the 3′UT. These primers could then beused to amplify a full-length hNPFF1 clone from human cDNA sources knownto express NPFF1 (for example, cerebellum, spinal cord, hippocampus,lung and kidney).

(SEQ ID NO: 17) BB609: 5′-CCACCCTTGTGGACAACCTCATCACTGGGTGGCCCTTCGACAATGCCACATGC-3′ (SEQ ID NO: 18) BB629: 5′-CTGCTCTGCATGGTGGGCAACACC-3′(SEQ ID NO: 19) A71: 5′-GACGGCGATGGTGACGAGCGC-3′Cloning of Human NPFF1 Receptor

The sequence of the human NPFF1 (hNPFF1) receptor from the initiatingmethionine to TMIV was determined to be present in a partial clone,plc29b, found in a Synaptic Pharmaceutical Corporation in-housedatabase. In order to isolate the full-length hNPFF1 receptor cDNA, ahuman cosmid library (Stratagene) was screened with a ³²P-labeled probe(BB609) corresponding to the II/III loop of plc29b. Partial DNAsequencing of one positive clone from this library, COS28a revealedsimilar sequence as had been previously shown for plc29b, with an introndownstream of TMIII. In order to obtain sequence in the 3′ end ofhNPFF1, COS28a was amplified with a vector primer and BB702, BB703 orBB704, forward primers in TMIV. DNA sequencing of these PCR productsresulted in the identification of TMIV through the stop codon.

Next, an in-house human spinal cord library was screened by PCR using aforward primer in the region of the initiating methionine (BB729) and areverse primer corresponding to TMIV (BB728). One positive pool, W4, wassubdivided and a positive sub-pool was screened by colony hybridizationwith a ³²P-labeled probe from TMII, BB676. Plasmid DNA was isolated forclone W4-18-4, renamed BO98, and DNA sequencing revealed that it wasfull-length but in the wrong orientation for expression in theexpression vector pEXJ. To obtain a full-length hNPFF1 construct in thecorrect orientation, BO98 was amplified with BB757, a forward primer atthe initiating methionine which contained an upstream BamHI site, andBB758, a reverse primer at the stop codon which contained a EcoRI site.The products from 3 independent PCR reactions were ligated intopcDNA3.1+ and transformed into DH5α cells. The sequence of one of thesetransformants, 3.3, was identical to the hNPFF1 sequence previouslydetermined from the consensus of BO98, COS28a and plc29b. Clone 3.3 wasrenamed BO102.

The hNPFF1 clone contains an open reading frame with 1293 nucleotides(see FIG. 7 and SEQ ID NO:7) and predicts a protein of 430 amino acids(FIG. 8 and SEQ ID NO:8). Hydrophobicity analysis reveals sevenhydrophobic domains which are presumed to be transmembrane domains (FIG.8). The sequence of hNPFF1 was determined to be most similar to the ratNPFF1 (86% nucleotide identity, 87% amino acid identity) and human NPFF2(56% nucleotide identity, 49% amino acid identity (FIG. 9)). The humanNPFF1 receptor also shares homology with human orexin₁ (53% nucleotideidentity, 35% amino acid identity), human orexin₂ (43% nucleotideidentity, 33% amino acid identity), human NPY₂ (47% nucleotide identity,31% amino acid identity), human CCK_(A) (46% nucleotide identity, 32%amino acid identity), and human CCK_(B) (46% nucleotide identity, 26%amino acid identity).

The following primers and probes were used in the cloning of hNPFF1:

(SEQ ID NO: 20) BB676: 5′-GTCACCAACATGTTCATCCTCAACCTGGCTGTCAGTGACCTGCTGGTGGGCATCTTCTGCATGCC-3′ (SEQ ID NO: 21) BB702:5′-GCGAGAAGCTGACCCTGCGGAAGG-3′ (SEQ ID NO: 22) BB703:5′-TCGTCACCATCGCCGTCATCTGGG-3′ (SEQ ID NO: 23) BB704:5′-CGTCATCTGGGCCGAGGGACACAG-3′ (SEQ ID NO: 24) BB728:5′-TGACGGCGATGGTGACGAGCGCC-3′ (SEQ ID NO: 25) BB729:5′-CAGCCTCCCAACAGCAGTTGGCC-3′ (SEQ ID NO: 26) BB757:5′-TAGCAAGGATCCGCATATGGAGGGGGAGCCCTCCC-3′ (SEQ ID NO: 27) BB758:5′-CTTCATGAATTCATCGCCTGCATGTATCTCGTGTCC-3′Cloning of Human NPFF2 ReceptorDiscovery of an Expressed Sequence Tag (EST) AA449919 in GENEMBLHomologous to rNPFF1 (hNPFF2)

A FASTA search of GENEMBL with the full-length sequence of rat NPFF1(rNPFF1) resulted in the identification of an EST (Accession numberAA449919) with a high degree of homology to. NPFF1 (57% identity at theDNA level). AA449919 is a 532 bp sequence annotated in Genbank as“Soares total fetus Nb2HF8 9w Homo sapiens cDNA clone 788698 5′ similarto SW:NYR_DROME P25931 NEUROPEPTIDE Y RECEPTOR,” which when translatedcorresponds to the region between the first extracellular loop and thebeginning of the sixth transmembrane domain of rNPFF1. GAP analysis ofAA449919 with rNPFF1 indicated that there is 57% DNA identity and a 50%amino acid identity between the two receptor sequences over this region.AA449919 displays 60% DNA identity and 59% amino acid identity over theregion that overlaps with the known sequence for hNPFF1 (firstextracellular loop to TM4), while over the same range rNPFF1 is 62% and61% identical to AA449919 at the DNA and amino acid levels,respectively. In comparison, hNPFF1 and rNPFF1 share 86% DNA identityand 92% amino acid identity over this region. Given the strong degree ofidentity between AA449919 and rNPFF1, AA449919 was given the nameNPFF-like (hNPFF2).

Cloning the Full-Length Sequence of (NPFF-Like) hNPFF2

To determine the full-length coding sequence of AA449919, 5′/3′ RapidAmplification of cDNA ends (RACE) was performed on Clontech Human SpleenMarathon-Ready cDNA (Clontech, Palo Alto, Calif.). For 5′ RACE, 5 μltemplate (human spleen Marathon-Ready cDNA was amplified witholigonucleotide primers JAB256 and AP1, the Expand Long DNA Template PCRSystem (Boehringer-Mannheim, Indianapolis, Ind.) and the following PCRprotocol were used: 94° C. hold for 3 minutes; 5 cycles of 94° C. for 30seconds, 72° C. for 4 minutes; 5 cycles of 94° C. for 30 seconds, 70° C.for 4 minutes; 30 cycles of 94° C. for 30 seconds, 68° C. for 4 minutes;68° C. hold for 4 minutes; 4° C. hold until products were ready to beloaded on a gel. 1 μl of this reaction was subjected to a second roundof amplification with primers JAB260 and AP2 and the same PCR protocol.For 3′ RACE, 5 μl human spleen Marathon-Ready cDNA was subjected to PCRwith primers JAB257 and AP1 with the same PCR protocol that was used for5′ RACE. 1 μl of this reaction was subjected to another round ofamplification using AP2 and JAB258 and the same PCR conditions.

The products were run on a 1% agarose TAE gel and bands greater than 500bp were extracted from the gel using the QIAQUICK gel extraction kit(QIAGEN, Chatsworth, Calif.). 5 μl of each purified band from the 5′ and3′ RACE reactions were directly sequenced with primers JAB261 (5′products) and JAB259 (3′ products) using the ABI Big Dye cyclesequencing protocol and ABI377 sequencers (ABI, Foster City, Calif.).The Wisconsin Package (GCG, Genetics Computer Group, Madison, Wis.) andSequencer 3.0 (Gene Codes Corporation, Ann Arbor, Mich.) were used toput together the full-length contiguous sequence of hNPFF2 from theAA449919 EST and the RACE products.

To attain the full-length hNPFF-like (hNPFF2) coding sequence forexpression, human spinal cord cDNA was amplified in eight independentPCR reactions using the Expand Long Template PCR System with buffer I(four of the eight reactions) or buffer 3 (4 reactions) and twooligonucleotide primers with restriction sites incorporated into their5′ ends: BB675 is a forward primer upstream of the initiating methionineand contains a BamHI site, and BB663. The PCR conditions for thisreaction were as follows: 94° C. hold for 5 minutes; 37 cycles of 94° C.for 30 seconds, 64° C. for 30 seconds, 68° C. for 2 minutes; a 7 minutehold at 68° C., and a 4° C. hold until products were ready to be loadedon a gel. The products were electrophoresed on a 1% agarose TAE gel, anda band of approximately 1.35 kb was cut and purified using the QIAQUICKgel extraction kit. The purified bands of seven of the eight reactionswere cut with BamHI and EcoRI, gel purified again using the same method,and ligated into pcDNA3.1(+) (Invitrogen, Carlsbad, Calif.). Eighteencolonies from the subsequent transformations were picked and determinedto be positive for NPFF-like by PCR. Eight of these 18 clones were fullysequenced, and one of these., BO89, was determined to be a full lengthclone with no point mutations. This construct was renamedpcDNA3.1-hNPFF2b.

For expression of NPFF-like in oocytes, one ul of each of these eightligations of the BB675-BB663 PCR product into pcDNA3.1(+) was subjectedto PCR with AN35, a pcDNA3.1 primer at the CMV promoter site, and the 3′NPFF-like primer BB663 using the Expand Long Template PCR System and thefollowing PCR protocol: 94° C. hold for 3 minutes; 37 cycles of 94° C.for 30 seconds, 65° C. for 30 seconds, 68° C. for 2 minutes; a 7 minutehold at 68° C., and a 4° C. hold until products were ready for in vitrotranscription. Of the seven PCR reactions, six yielded products of theexpected size.

The following is a list of primers and their associated sequences whichwere used in the cloning of this receptor (hNPFF2):

(SEQ ID NO: 28) AN35: 5′-CGTGTACGGTGGGAGGTCTATATAAGCAGAG-3′ (SEQ ID NO:29) AP1: 5′-CCATCCTAATACGACTCACTATAGGGC-3′ (SEQ ID NO: 30) AP2:5′-ACTCACTATAGGGCTCGAGCGGC-3′ (SEQ ID NO: 31) JAB256:5′-TGATAGTGAGCTTTGGTTTAAAAGGG-3′ (SEQ ID NO: 32) JAB257:5′-GAAGATCTACACCACTGTGCTGTTTG-3′ (SEQ ID NO: 33) JAB258:5′-AACATCTACCTGGCTCCCCTCTCCC-3′ (SEQ ID NO: 34) JAB259:5′-TTGTCATCATGTATGGAAGGATTGG-3′ (SEQ ID NO: 35) JAB260:5′-GACCACACACTGGAACCTATCTAC-3′ (SEQ ID NO: 36) JAB261:5′-GCAATTGCAACTAACGTAAAGACTG-3′ (SEQ ID NO: 37) BB675:5′-TAGCAAGGATCCGAGGTTCATCATGAATGAGAAATGG- 3′ (SEQ ID NO: 38) BB663:5′-CTTCATGAATTCGCGTAGTAGAGTTAGGATTATCAC- 3′

For expression of NPFF2, mRNA transcripts were generated as describedfor NPFF1, using PCR products from ligation reactions or linearized DNAfrom BO89 as DNA templates. Oocytes were injected with 5-50 ng NPFF2mRNA and incubated as previously described.

Isolation of the Rat Homologue of NPFF2

To obtain a fragment of the rat homologue of NPFF2, rat genomic DNA(Clontech, Palo Alto, Calif.), rat hypothalamic cDNA or rat spinal cordcDNA was amplified with a forward PCR primer corresponding to TMIV ofhuman NPFF2 (JAB307) and a reverse primer corresponding to TMVI of humanNPFF2 (JAB 306). PCR was performed with the Expand Long Template PCRSystem (Roche Molecular Biochemicals, Indianapolis, Ind.) under thefollowing conditions: 1 minute at 94° C., 2 minutes at 50° C., 2 minutesat 68° C. for 40 cycles, with a pre- and post-incubation of 3 minutes at94° C. and 4 minutes at 68° C. respectively. Bands of 368 bp from 3independent PCR reactions were isolated from a TAE gel, purified usingthe QIAQUICK gel extraction kit (QIAGEN, Chatsworth, Calif.), andsequenced on both strands as described above. The sequences of these 3PCR products were identical.

To obtain additional sequence for rat NPFF2, reduced stringency PCR wasperformed using primers designed against the human NPFF2 NH₂ and COOHtermini along with PCR primers designed against the rat NPFF2 fragment.For the NH₂ terminal sequence, PCR was performed on rat spinal cord cDNAwith BB665, a sense primer just upstream of TMI in human NPFF2, andBB795, an antisense primer in the second extracellular loop of the ratNPFF2. For the COOH terminal sequence, PCR was performed on rat spinalcord cDNA with BB793, a sense primer from the third intracellular loopin rat NPFF2, and BB668, an antisense primer just downstream from TMVIIin human NPFF2. PCR was performed using the Expand Long Template PCRSystem (Roche Biochemicals, Indianapolis, Ind.) with buffer 2 (NH₂terminal) or buffer 1 (COOH terminal) and the following conditions: 30seconds at 94° C., 30 seconds at 42° C. (NH₂ terminal) or 50° C. (COOHterminal), 1.5 minutes at 68° C. for 40 cycles, with a pre- andpost-incubation of 3 minutes at 94° C. and 4 minutes at 68° C.respectively. A 500 bp band from the NH₂ terminal PCR and a 300 bp bandfrom the COOH terminal PCR were isolated from a TAE gel, purified usingthe QIAQUICK gel extraction kit (QIAGEN, Chatsworth, Calif.), andsequenced on both strands as described above.

A rat liver genomic phage library (2.75 million recombinants,Stratagene, LaJolla, Calif.) was screened using a ³²P-labeledoligonucleotide probe, BB712, corresponding to the second extracellularloop and TMV of the rat NPFF2 fragment above. Hybridization ofnitrocellulose filter overlays of the plates was performed at highstringency: 42° C. in a solution containing 50% formamide, 5×SSC (1×SSCis 0.15M sodium chloride, 0.015M sodium citrate), 1× Denhardt's solution(0.02% polyvinylpyrrolindone, 0.02% Ficoll, 0.02% bovine serum albumin),7 mM Tris and 25 μg/ml sonicated salmon sperm DNA. The filters werewashed at 55° C. in 0.1×SSC containing 0.1% sodium dodecyl sulfate andexposed at −70° C. to Kodak BioMax MS film in the presence of anintensifying screen.

Three positive signals, rNPFF2-1, rNPFF2-4 and rNPFF2-6 were isolated ona tertiary plating. A 3.5 kb fragment, from a BglII/EcoRI digest of DNAisolated from rNPFF2-6, was identified by Southern blot analysis withBB712, subcloned into pcDNA3.1 (Invitrogen, San Diego, Calif.) and usedto transform E. coli DH5α cells (Gibco BRL, Gaithersburg Md.). PlasmidDNA from one transformant was sequenced using an ABI 377 sequencer asdescribed above. Sequencing with HK137, a sense primer from TMV of therat NPFF2 fragment revealed the sequence for TMVII, the COOH terminusand some 3′UT. Sequencing with HK139, an antisense primer from TMII ofrNPFF2, revealed the presence an intron upstream of TMII.

To determine if any of the three positive plaques contained sequenceupstream of this intron, DNA from each of the clones were spotted ontonitrocellulose and hybridized with HK140, an oligonucleotide probecorresponding to TMI of the rat NPFF2 fragment. The rNPFF2-1 andrNPFF2-4 clones were positive. A 2.1 kb fragment, from a HindIII digestof DNA isolated from rNPFF2-4, was identified by Southern blot analysiswith HK140, subcloned into pcDNA3.1 (Invitrogen, San Diego, Calif.) andused to transform E. coli DH5α cells (Gibco BRL, Gaithersburg Md.).Sequencing of this fragment with HK138, an antisense primer from TMI ofrat NPFF2, revealed the NH₂ terminus and 5′UT of the rat NPFF2 receptor.

The full-length NPFF2 was amplified from rat spinal cord cDNA using asense primer in the 5′UT (HK146, also incorporating a BamHI restrictionsite) and an antisense primer from the 3′UT (HK147, also incorporating aBstXI restriction site) and the Expand Long Template PCR System (RocheMolecular Biochemicals, Indianapolis, Ind.) using buffer 2 and thefollowing PCR conditions: 30 seconds at 94° C., 2.5 minutes at 68° C.for 32 cycles, with a pre- and post-incubation of 5 minutes at 94° C.and 7 minutes at 68° C., respectively. Products from 5 independent PCRreactions were gel-purified. 1 μl of each reaction was used as atemplate to re-amplify the product using the same PCR conditions. Theproducts were digested with BamHI and BstXI and ligated into a modifiedpcDNA3.1 vector (Invitrogen, San Diego, Calif.). Products from each PCRreaction were sequenced as above. While a consensus amino acid sequencewas determined among the PCR products, there was some ambiguity in thenucleotide sequence at 4 positions. To determine if this representedPCR-induced errors or allelic variations, the areas in question wereamplified from several lots of genomic DNA. Sequencing of these genomicproducts revealed the same ambiguities, suggesting allelic variations atthese residues. One construct, KO31, was renamed BO119 and later renamedpcDNA3.1-rNPFF2-f.

Oligonucleotide primers and probes used in the identification andisolation of the rat NPFF2:

(SEQ ID NO: 47) JAB307: 5′-TTTGTCATTATTATGATCATCTGG-3′ (SEQ ID NO: 48)JAB306: 5′-AATAAAAAGCAGGGCCACAATCAG-3′ (SEQ ID NO: 49) BB665:5′-TCATTATTTCCTACTTTCTGATC-3′ (SEQ ID NO: 50) BB795:5′-CTCATTTCCTGGTTTGGCCAATCC-3′ (SEQ ID NO: 51) BB793:5′-TCTTCAAGACCTCAGCACACAGC-3′ (SEQ ID NO: 52) BB668:5′-GAGCTGGAAAGCTTCTTGGAAACC-3′ (SEQ ID NO: 53) BB712:5′-CTGGTGTCGGGAGGATTGGCCAAACCAGGAAATGAGGAG GATCTACACC-3′ (SEQ ID NO: 54)HK137: 5′-GCAGTGTCAACCCCATCATTTATGG-3′ (SEQ ID NO: 55) HK138:5′-CAAAGCAAACGACAGTGTTTCCCACC-3′ (SEQ ID NO: 56) HK139:5′-AGTGACCGTGTGCATGTACCTATTCC-3′ (SEQ ID NO: 57) HK140:5′-GGTGGGAAACACTGTCGTTTGCTTTGTTGTAATAAGGAA TAGGTACATGCACACGGTCAC-3′ (SEQID NO: 58) HK146: 5′-GTCACGGATCCAGCCTCTCCTTTGATAAGGTCCACC-3′ (SEQ ID NO:59) HK147: 5′-GTCAGCCATCGAGTTGGCTTCGTATGCTATATAACA TTGGATAGC-3′Isolation of Other Species Homologs of NPFF1 or NPFF2 Receptor cDNA

A nucleic acid sequence encoding a NPFF1 or NPFF2 receptor cDNA fromother species may be isolated using standard molecular biologytechniques and approaches such as those described below:

Approach #1: A genomic library (e.g., cosmid, phage, P1, BAC, YAC)generated from the species of interest may be screened with a³²P-labeled oligonucleotide probe corresponding to a fragment of thehuman or rat NPFF1 or NPFF2 receptors whose sequence is shown in SEQ IDNOs:1, 5, 7 and 43 to isolate a genomic clone. The full-length sequencemay be obtained by sequencing this genomic clone. If one or more intronsare present in the gene, the full-length intronless gene may be obtainedfrom cDNA using standard molecular biology techniques. For example, aforward PCR primer designed in the 5′UT and a reverse PCR primerdesigned in the 3′UT may be used to amplify a full-length, intronlessreceptor from cDNA. Standard molecular biology techniques could be usedto subclone this gene into a mammalian expression vector.

Approach #2: Standard molecular biology techniques may be used to screencommercial cDNA phage libraries of the species of interest byhybridization under reduced stringency with a ³²P-labeledoligonucleotide probe corresponding to a fragment of the sequences shownin SEQ ID NOs:1, 5, 7 and 43. One may isolate a full-length NPFF1 orNPFF2 receptor by obtaining a plaque purified clone from the lambdalibraries and then subjecting the clone to direct DNA sequencing.Alternatively, standard molecular biology techniques could be used toscreen cDNA plasmid libraries by PCR amplification of library poolsusing primers designed against a partial species homolog sequence. Afull-length clone may be isolated by Southern hybridization of colonylifts of positive pools with a ³²P-labeled oligonucleotide probe.

Approach #3: 3′ and 5′ RACE may be utilized to generate PCR productsfrom cDNA derived from the species of interest expressing NPFF1 or NPFF2which contain the additional sequence of NPFF1 or NPFF2. These RACE PCRproducts may then be sequenced to determine the additional sequence.This new sequence is then used to design a forward PCR primer in the5′UT and a reverse primer in the 3′UT. These primers are then used toamplify a full-length NPFF1 or NPFF2 clone from cDNA.

Examples of other species include, but are not limited to, mouse, dog,monkey, hamster and guinea pig.

Cell Culture

COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days.

Human embryonic kidney 293 cells (HEK-293 cells) are grown on 150 mmplates in DMEM with supplements (10% bovine calf serum, 4 mM glutamine,100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂. Stockplates of 293 cells are trypsinized and split 1:6 every 3-4 days.

Mouse fibroblast LM(tk−) cells are grown on 150 mm plates in D-MEM withsupplements (Dulbecco's Modified Eagle Medium with 10% bovine calfserum, 4 mM glutamine, 100 units/ml penicillin/100 μg/ml streptomycin)at 37° C., 5% CO₂. Stock plates of LM(tk−) cells are trypsinized andsplit 1:10 every 3-4 days.

Chinese hamster ovary (CHO) cells were grown on 150 mm plates in HAM'sF-12 medium with supplements (10% bovine calf serum, 4 mM L-glutamineand 100 units/ml penicillin/100 ug/ml streptomycin) at 37° C., 5% CO₂.Stock plates of CHO cells are trypsinized and split 1:8 every 3-4 days.

Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells aretrypsinized and split 1:15 every 3-4 days.

Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue culturedishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C., no CO₂. High Five insect cells are grown on 150 mm tissue culturedishes in Ex-Cell 400™ medium supplemented with L-Glutamine, also at 27°C., no CO₂.

Transient Transfection

Receptors studied may be transiently transfected into COS-7 cells by theDEAE-dextran method using 1 μg of DNA/10⁶ cells (Cullen, 1987). Inaddition, Schneider 2 Drosophila cells may be cotransfected with vectorscontaining the receptor gene under control of a promoter which is activein insect cells, and a selectable resistance gene, eg., the G418resistant neomycin gene, for expression of the polypeptides disclosedherein.

Stable Transfection

DNA encoding the human receptor disclosed herein may be co-transfectedwith a G-418 resistant gene into the human embryonic kidney 293 cellline by a calcium phosphate transfection method (Cullen, 1987). Stablytransfected cells are selected with G-418.

Membrane Preparations

LM(tk−) cells stably transfected with the DNA encoding the humanreceptor disclosed herein may be routinely converted from an adherentmonolayer to a viable suspension. Adherent cells are harvested withtrypsin at the point of confluence, resuspended in a minimal volume ofcomplete DMEM for a cell count, and further diluted to a concentrationof 10⁶ cells/ml in suspension media (10% bovine calf serum, 10% 10×Medium 199 (Gibco), 9 mM NaHCO₃, 25 mM glucose, 2 mM L-glutamine, 100units/ml penicillin/100 μg/ml streptomycin, and 0.05% methyl cellulose).Cell suspensions are maintained in a shaking incubator at 37° C., 5% CO₂for 24 hours. Membranes harvested from cells grown in this manner may bestored as large, uniform batches in liquid nitrogen. Alternatively,cells may be returned to adherent cell culture in complete DMEM bydistribution into 96-well microtiter plates coated with poly-D-lysine(0.01 mg/ml) followed by incubation at 37° C., 5% CO₂ for 24 hours.

Generation of Baculovirus

The coding region of DNA encoding the human receptors disclosed hereinmay be subcloned into pBlueBacIII into existing restriction sites orsites engineered into sequences 5′ and 3′ to the coding region of thepolypeptides. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of DNA construct encoding a polypeptide may be co-transfectedinto 2×10⁶ Spodoptera frugiperda insect Sf9 cells by the calciumphosphate co-precipitation method, as outlined by Pharmingen (in“Baculovirus Expression Vector System: Procedures and Methods Manual”).The cells then are incubated for 5 days at 27° C.

The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

Radioligand Binding Assays

Cells may be screened for the presence of endogenous human receptorusing radioligand binding or functional assays (described in detail inthe following experimental description). Cells with either no or a lowlevel of the endogenous human receptors disclosed herein present may betransfected with the human receptors.

Transfected cells from culture flasks are scraped into 5 ml of 20 mMTris-HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. The cell lysatesare centrifuged at 1000 rpm for 5 min. at 4° C., and the supernatant iscentrifuged at 30,000×g for 20 min. at 4° C. The pellet is suspended inbinding buffer (50 mM Tris-HCl, 60 mM NaCl, 1 mM MgCl, 33 μM EDTA, 33 μMEGTA at pH 7.4 supplemented with 0.2% BSA, 2 μg/ml aprotinin, and 20 μMbestatin). Optimal membrane suspension dilutions, defined as the proteinconcentration required to bind less than 10% of the added radioligand,are added to 96-well polpropylene microtiter plates containing³H-labeled compound, unlabeled compounds, and binding buffer to a finalvolume of 250 μl. In equilibrium saturation binding assays membranepreparations are incubated in the presence of increasing concentrationsof [³H]-labeled compound. The binding affinities of the differentcompounds are determined in equilibrium competition binding assays,using [¹²⁵I]-labeled compound in the presence of ten to twelve differentconcentrations of the displacing ligands. Competition assay: 50 pMradioligand, 10-12 points. Binding reaction mixtures are incubated for 2hr at 25° C., and the reaction stopped by filtration through a doublelayer of GF filters treated with 0.1% polyethyleneimine, using a cellharvester. Wash buffer: 50 mM Tris-HCl, 0.1% BSA. Radioactivity may bemeasured by scintillation counting and data are analyzed by acomputerized non-linear regression program. Non-specific binding isdefined as the amount of radioactivity remaining after incubation ofmembrane protein in the presence of 1 μM final concentration unlabeled.Protein concentration may be measured by the Bradford method usingBio-Rad Reagent, with bovine serum albumin as a standard.

Functional Assays

Cells may be screened for the presence of endogenous mammalian receptorusing radioligand binding or functional assays (described in detail inthe above or following experimental description, respectively). Cellswith no or a low level of endogenous receptor present may be transfectedwith the mammalian receptor for use in the following functional assays.

A wide spectrum of assays can be employed to screen for the presence ofreceptor ligands. These range from traditional measurements ofphosphatidyl inositol, cAMP, Ca⁺⁺, and K⁺, for example; to systemsmeasuring these same second messengers but which have been modified oradapted to be higher throughput, more generic, and more sensitive; tocell based platforms reporting more general cellular events resultingfrom receptor activation such as metabolic changes, differentiation, andcell division/proliferation, for example; to high level organism assayswhich monitor complex physiological or behavioral changes thought to beinvolved with receptor activation including cardiovascular, analgesic,orexigenic, anxiolytic, and sedation effects, for example.

Cyclic AMP (cAMP) Formation Assay

The receptor-mediated inhibition of cyclic AMP (cAMP) formation may beassayed in transfected cells expressing the mammalian receptors. Cellsare plated in 96-well plates and incubated in Dulbecco's phosphatebuffered saline (PBS) supplemented with 10 mM HEPES, 5 mM theophylline,2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon for20 min at 37° C., in 5% CO₂. Test compounds are added and incubated foran additional 10 min at 37° C. The medium is then aspirated and thereaction stopped by the addition of 100 mM HCl. The plates are stored at4° C. for 15 min, and the cAMP content in the stopping solution measuredby radioimmunoassay. Radioactivity may be quantified using a gammacounter equipped with data reduction software.

Arachidonic Acid Release Assay

Cells stably transfected with the mammalian receptor are seeded into 96well plates and grown for 3 days in HAM's F-12 with supplements.³H-arachidonic acid (specific activity=0.75 μCi/ml) is delivered as a100 μL aliquot to each well and samples were incubated at 37° C., 5% CO₂for 18 hours. The labeled cells are washed three times with 200 μL HAM'sF-12. The wells are then filled with medium (200 μL) and the assay isinitiated with the addition of peptides or buffer (22 μL). Cells areincubated for 30 min at 37° C., 5% CO₂. Supernatants are transferred toa microtiter plate and evaporated to dryness at 75° C. in a vacuum oven.Samples are then dissolved and resuspended in 25 μL distilled water.Scintillant (300 μL) is added to each well and samples are counted for³H in a Trilux plate reader. Data are analyzed using nonlinearregression and statistical techniques available in the GraphPAD Prismpackage (San Diego, Calif.).

Intracellular Calcium Mobilization Assay

The intracellular free calcium concentration may be measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(Bush et al, 1991). Stably transfected cells are seeded onto a 35 mmculture dish containing a glass coverslip insert. Cells are washed withHBS and loaded with 100 μL of Fura-2/AM (10 μM) for 20 to 40 min. Afterwashing with HBS to remove the Fura-2/AM solution, cells areequilibrated in HBS for 10 to 20 min. Cells are then visualized underthe 40× objective of a Leitz Fluovert FS microscope and fluorescenceemission is determined at 510 nM with excitation wavelengths alternatingbetween 340 nM and 380 nM. Raw fluorescence data are converted tocalcium concentrations using standard calcium concentration curves andsoftware analysis techniques.

Alternatively, cells expressing the mammalian receptor DNA are plated in96-well plates and grown to confluence. Cells are incubated with a cellpermeant fluorescent calcium indicator such as, but not restricted to,Fluo-3/AM. After washing with HBS to remove the Fluo-3/AM solution,cells are equilibrated for 20 min. The fluorescence emission due tointracellular calcium mobilization elicited by agonists of the expressedmammalian receptor, is determined with a fluorescence imaging platereader (FLIPR, Molecular Devices Corporation, Sunnyvale, Calif.).

Phosphoinositide Metabolism Assay

Cells stably expressing the mammalian receptor cDNA are plated in96-well plates and grown to confluence. The day before the assay thegrowth medium is changed to 100 μl of medium containing 1% serum and 0.5μCi [³H]myo-inositol, and the plates are incubated overnight in a CO₂incubator (5% CO₂ at 37° C.). Alternatively, arachidonic acid releasemay be measured if [³H]arachidonic acid is substituted for the[³H]myo-inositol. Immediately before the assay, the medium is removedand replaced by 200 μL of PBS containing 10 mM LiCl, and the cells areequilibrated with the new medium for 20 min. During this interval cellsare also equilibrated with the antagonist, added as a 10 μL aliquot of a20-fold concentrated solution in PBS. The [³H]inositol-phosphatesaccumulation from inositol phospholipid metabolism may be started byadding 10 μL of a solution containing the agonist. To the first well 10μL may be added to measure basal accumulation, and 11 differentconcentrations of agonist are assayed in the following 11 wells of eachplate row. All assays are performed in duplicate by repeating the sameadditions in two consecutive plate rows. The plates are incubated in aCO₂ incubator for 1 hr. The reaction may be terminated by adding 15 μLof 50% v/v trichloroacetic acid (TCA), followed by a 40 min. incubationat 4° C. After neutralizing TCA with 40 μL of 1 M Tris, the content ofthe wells may be transferred to a Multiscreen HV filter plate(Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form). Thefilter plates are prepared adding 200 μL of Dowex AG1-X8 suspension (50%v/v, water: resin) to each well. The filter plates are placed on avacuum manifold to wash or elute the resin bed. Each well is washed 2times with 200 μL of water, followed by 2×200 μL of 5 mM sodiumtetraborate/60 mM ammonium formate. The [³H]IPs are eluted into empty96-well plates with 200 μL of 1.2 M ammonium formate/0.1 formic acid.The content of the wells is added to 3 ml of scintillation cocktail, andthe radioactivity is determined by liquid scintillation counting.

GTPγS Functional Assay

Membranes from cells transfected with the mammalian receptors aresuspended in assay buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.4)supplemented with 0.1% BSA, 0.1% bacitracin and 10 μM GDP. Membranes areincubated on ice for 20 minutes, transferred to a 96-well Milliporemicrotiter GF/C filter plate and mixed with GTPγ³⁵S (e.g., 250,000cpm/sample, specific activity ^(˜)1000 Ci/mmol) plus or minus GTPγS(final concentration=100 μM). Final membrane protein concentration≈90μg/ml. Samples are incubated in the presence or absence of porcinegalanin (final concentration=1 μM) for 30 min. at room temperature, thenfiltered on a Millipore vacuum manifold and washed three times with coldassay buffer. Samples collected in the filter plate are treated withscintillant and counted for ³⁵S in a Trilux (Wallac) liquidscintillation counter. It is expected that optimal results are obtainedwhen the mammalian receptor membrane preparation is derived from anappropriately engineered heterologous expression system, i.e., anexpression system resulting in high levels of expression of themammalian receptor and/or expressing G-proteins having high turnoverrates (for the exchange of GDP for GTP). GTPγS assays are well-known inthe art, and it is expected that variations on the method describedabove, such as are described by e.g., Tian et al. (1994) or Lazareno andBirdsall (1993), may be used by one of ordinary skill in the art.

MAP Kinase Assay

MAP kinase (mitogen activated kinase) may be monitored to evaluatereceptor activation. MAP kinase is activated by multiple pathways in thecell. A primary mode of activation involves the ras/raf/MEK/MAP kinasepathway. Growth factor (tyrosine kinase) receptors feed into thispathway via SHC/Grb-2/SOS/ras. Gi coupled receptors are also known toactivate ras and subsequently produce an activation of MAP kinase.Receptors that activate phospholipase C (Gq and G11) producediacylglycerol (DAG) as a consequence of phosphatidyl inositolhydrolysis. DAG activates protein kinase C which in turn phosphorylatesMAP kinase.

MAP kinase activation can be detected by several approaches. Oneapproach is based on an evaluation of the phosphorylation state, eitherunphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the mitogen and then extracted with Laemmli buffer. Thesoluble fraction is applied to an SDS-PAGE gel and proteins aretransferred electrophoretically to nitrocellulose or Immobilon.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

Another approach is based on evaluation of the MAP kinase activity via aphosphorylation assay. Cells are stimulated with the mitogen and asoluble extract is prepared. The extract is incubated at 30° C. for 10min with gamma-³²P-ATP, an ATP regenerating system, and a specificsubstrate for MAP kinase such as phosphorylated heat and acid stableprotein regulated by insulin, or PHAS-I. The reaction is terminated bythe addition of H₃PO₄ and samples are transferred to ice. An aliquot isspotted onto Whatman P81 chromatography paper, which retains thephosphorylated protein. The chromatography paper is washed and countedfor ³²P in a liquid scintillation counter. Alternatively, the cellextract is incubated with gamma-³²P-ATP, an ATP regenerating system, andbiotinylated myelin basic protein bound by streptavidin to a filtersupport. The myelin basic protein is a substrate for activated MAPkinase. The phosphorylation reaction is carried out for 10 min at 30° C.The extract can then by aspirated through the filter, which retains thephosphorylated myelin basic protein. The filter is washed and countedfor ³²P by liquid scintillation counting.

Cell Proliferation Assay

Receptor activation of a G protein coupled receptor may lead to amitogenic or proliferative response which can be monitored via³H-thymidine uptake. When cultured cells are incubated with³H-thymidine, the thymidine translocates into the nuclei where it isphosphorylated to thymidine triphosphate. The nucleotide triphosphate isthen incorporated into the cellular DNA at a rate that is proportionalto the rate of cell growth. Typically, cells are grown in culture for1-3 days. Cells are forced into quiescence by the removal of serum for24 hrs. A mitogenic agent is then added to the media. 24 hrs later, thecells are incubated with ³H-thymidine at specific activities rangingfrom 1 to 10 uCi/ml for 2-6 hrs. Harvesting procedures may involvetrypsinization and trapping of cells by filtration over GF/C filterswith or without a prior incubation in TCA to extract soluble thymidine.The filters are processed with scintillant and counted for ³H by liquidscintillation counting. Alternatively, adherant cells are fixed in MeOHor TCA, washed in water, and solubilized in 0.05% deoxycholate/0.1 NNaOH. The soluble extract is transferred to scintillation vials andcounted for ³H by liquid scintillation counting.

Promiscuous Second Messenger Assays

It is possible to coax receptors of different functional classes tosignal through a pre-selected pathway through the use of promiscuousG_(α) subunits. For example, by providing a cell based receptor assaysystem with an endogenously supplied promiscuous G_(α) subunit such asG_(α16) or a chimeric G_(α) subunit such as G_(αzq), a GPCR, which mightnormally prefer to couple through a specific signaling pathway (e.g.,G_(g), G_(i), G_(q), G₀, etc.), can be made to couple through thepathway defined by the promiscuous G_(α) subunit and upon agonistactivation produce the second messenger associated with that subunit'spathway. In the case of G_(α16) and/or G_(αqz) this would involveactivation of the G_(q) pathway and production of the second messengerphosphotidyl inositol. Through the use of similar strategies and tools,it is possible to bias receptor signaling through pathways producingother second messengers such as Ca⁺⁺, cAMP, and K⁺ currents, forexample.

Microphysiometric Measurement of Receptor Mediated ExtracellularAcidification Rates

Because cellular metabolism is intricately involved in a broad range ofcellular events (including receptor activation of multiple messengerpathways), the use of microphysiometric measurements of cell metabolismcan in principle provide a generic assay of cellular activity arisingfrom the activation of any receptor regardless of the specifics of thereceptor's signaling pathway.

General guidelines for transient receptor expression, cell preparationand microphysiometric recording are described elsewhere (Salon, J. A.and Owicki, J. A., 1996). Receptors and/or control vectors aretransiently expressed in CHO-K1 cells, by liposome mediated transfectionaccording to the manufacturers recommendations (LipofectAMINE, GibcoBRL,Gaithersburg, Md.), and maintained in Ham's F-12 complete (10% serum). Atotal of 10 g of DNA is used to transfect each 75 cm² flask which hadbeen split 24 hours prior to the transfection and judged to be 70-80%confluent at the time of transfection. 24 hours post transfection, thecells are harvested and 3×10⁵ cells seeded into microphysiometercapsules. Cells are allowed to attach to the capsule membrane for anadditional 24 hours; during the last 16 hours, the cells are switched toserum-free F-12 complete to minimize ill-defined metabolic stimulationcaused by assorted serum factors. On the day of the experiment the cellcapsules are transferred to the microphysiometer and allowed toequilibrate in recording media (low buffer RPMI 1640, no bicarbonate, noserum (Molecular Devices Corporation, Sunnyvale, Calif.) containing 0.1%fatty acid free BSA), during which a baseline measurement of basalmetabolic activity is established.

A standard recording protocol specifies a 100 μl/min flow rate, with a 2min total pump cycle which includes a 30 sec flow interruption duringwhich the acidification rate measurement is taken. Ligand challengesinvolve a 1 min 20 sec exposure to the sample just prior to the firstpost challenge rate measurement being taken, followed by two additionalpump cycles for a total of 5 min 20 sec sample exposure. Typically,drugs in a primary screen are presented to the cells at 10 μM finalconcentration. Follow up experiments to examine dose-dependency ofactive compounds is then done by sequentially challenging he cells witha drug concentration range that exceeds the amount needed to generateresponses ranging from threshold to maximal levels. Peptides included inthe microphysiometric screen included rat NPFF (FLFQPQRF-NH2) (SEQ IDNO: 45) and rat A-18-F-amide (AGEGLSSPFWSLAAPQRF-NH2) (SEQ ID NO: 46).Ligand samples are then washed out and the acidification rates reportedare expressed as a percentage increase of the peak response over thebaseline rate observed just prior to challenge.

Receptor/G Protein co-Transfection Studies

A strategy for determining whether NPFF can couple preferentially toselected G proteins involves co-transfection of NPFF receptor cDNA intoa host cell together with the cDNA for a G protein alpha sub-unit.Examples of G alpha sub-units include members of the Gαi/Gαo class(including Gαt2 and Gαz), the Gαq class, the Gas class, and the Gα12/13class. A typical procedure involves transient transfection into a hostcell such as COS-7. Other host cells may be used. A key consideration iswhether the cell has a downstream effector (a particular adenylatecyclase, phospholipase C, or channel isoform, for example) to support afunctional response through the G protein under investigation. G proteinbeta gamma sub-units native to the cell are presumed to complete the Gprotein heterotrimer; otherwise specific beta and gamma sub-units may beco-transfected as well. Additionally, any individual or combination ofalpha, beta, or gamma subunits may be co-transfected to optimize thefunctional signal mediated by the receptor.

The receptor/G alpha co-transfected cells are evaluated in a bindingassay, in which case the radioligand binding may be enhanced by thepresence of the optimal G protein coupling or in a functional assaydesigned to test the receptor/G protein hypothesis. In one example, theNPFF receptor may be hypothesized to inhibit cAMP accumulation throughcoupling with G alpha sub-units of the Gαi/Gαo class. Host cellsco-transfected with the NPFF receptor and appropriate G alpha sub-unitcDNA are stimulated with forskolin +/− NPFF agonist, as described abovein cAMP methods. Intracellular cAMP is extracted for analysis byradioimmunoassay. Other assays may be substituted for cAMP inhibition,including GTPγ³⁵S binding assays and inositol phosphate hydrolysisassays. Host cells transfected with NPFF minus G alpha or with G alphaminus NPFF would be tested simultaneously as negative controls. NPFFreceptor expression in transfected cells may be confirmed in ¹²⁵I-NPFFprotein binding studies using membranes from transfected cells. G alphaexpression in transfected cells may be confirmed by Western blotanalysis of membranes from transfected cells, using antibodies specificfor the G protein of interest.

The efficiency of the transient transfection procedure is a criticalfactor for signal to noise in an inhibitory assay, much more so than ina stimulatory assay. If a positive signal present in all cells (such asforskolin-stimulated cAMP accumulation) is inhibited only in thefraction of cells successfully transfected with receptor and G alpha,the signal to noise ratio will be poor. One method for improving thesignal to noise ratio is to create a stably transfected cell line inwhich 100% of the cells express both the receptor and the G alphasubunit. Another method involves transient co-transfection with a thirdcDNA for a G protein-coupled receptor which positively regulates thesignal which is to be inhibited. If the co-transfected cellssimultaneously express the stimulatory receptor, the inhibitoryreceptor, and a requisite G protein for the inhibitory receptor, then apositive signal may be elevated selectively in transfected cells using areceptor-specific agonist. An example involves co-transfection of COS-7cells with 5-HT4, NPFF1, and a G alpha sub-unit. Transfected cells arestimulated with a 5-HT4 agonist +/− NPFF1 protein. Cyclic AMP isexpected to be elevated only in the cells also expressing NPFF1 and theG alpha subunit of interest, and a NPFF-dependent inhibition may bemeasured with an improved signal to noise ratio.

It is to be understood that the cell lines described herein are merelyillustrative of the methods used to evaluate the binding and function ofthe mammalian receptors of the present invention, and that othersuitable cells may be used in the assays described herein.

Electrophysiology

Methods for Recording Currents in Xenopus oocytes

Oocytes were harvested from Xenopus laevis and injected with mRNAtranscripts as previously described (Quick and Lester, 1994; Smith etal., 1997). NPFF receptors and Gα_(q/z) chimera synthetic RNAtranscripts were synthesized using the T7 polymerase (“Message Machine,”Ambion) from linearized plasmids or PCR products containing the completecoding region of the genes. Oocytes were injected with 10 ng NPFFreceptors synthetic RNA and incubated for 3-8 days at 17 degrees. Threeto eight hours prior to recording, oocytes were injected with 500 pgGα_(q/z) mRNA in order to observe coupling to Ca⁺⁺ activated Cl⁻currents. Dual electrode voltage clamp (Axon Instruments Inc.) wasperformed using 3 M KCl-filled glass microelectrodes having resistancesof 1-2 Mohm. Unless otherwise specified, oocytes were voltage clamped ata holding potential of −80 mV. During recordings, oocytes were bathed incontinuously flowing (1-3 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5 (ND96). Drugs wereapplied either by local perfusion from a 10 ml glass capillary tubefixed at a distance of 0.5 mm from the oocyte, or by switching from aseries of gravity fed perfusion lines.

Other oocytes may be injected with a mixture of receptor mRNAs andsynthetic mRNA encoding the genes for G-protein-activated inwardrectifiers (GIRK1 and GIRK4, U.S. Pat. Nos. 5,734,021 and 5,728,535).Genes encoding G-protein inwardly rectifying K⁺ (GIRK) channels 1 and 4(GIRK1 and GIRK4) may be obtained by PCR using the published sequences(Kubo et al., 1993; Dascal et al., 1993; Krapivinsky et al., 1995 and1995b) to derive appropriate 5′ and 3′ primers. Human heart cDNA may beused as template together with appropriate primers.

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen et al., 1983; Takahashi et al., 1987). Activationof the phospholipase C (PLC) pathway is assayed by applying testcompound in ND96 solution to oocytes previously injected with mRNA forthe mammalian receptor and observing inward currents at a holdingpotential of −80 mV. The appearance of currents that reverse at −25 mVand display other properties of the Ca⁺⁺-activated Cl⁻ (chloride)channel is indicative of mammalian receptor-activation of PLC andrelease of IP3 and intracellular Ca⁺⁺. Such activity is exhibited byGPCRs that couple to G_(q).

Measurement of inwardly rectifying K⁺ (potassium) channel (GIRK)activity may be monitored in oocytes that have been co-injected withmRNAs encoding the mammalian receptor, GIRK1, and GIRK4. The two GIRKgene products co-assemble to form a G-protein activated potassiumchannel known to be activated (i.e., stimulated) by a number of GPCRsthat couple to G_(i) or G_(o) (Kubo et al., 1993; Dascal et al., 1993).Oocytes expressing the mammalian receptor plus the two GIRK subunits aretested for test compound responsivity by measuring K⁺ currents inelevated K⁺ solution containing 49 mM K⁺. Activation of inwardlyrectifying currents that are sensitive to 300 μM Ba⁺⁺ signifies themammalian receptor coupling to a G_(i) or G_(o) pathway in the oocytes.

Localization of mRNA Coding for Rat NPFF1 Receptors

Development of probes for NPFF1: To facilitate the production ofradiolabeled, antisense RNA probes a fragment of the gene encoding ratNPFF1 was subcloned into a plasmid vector containing RNA polymerasepromotor sites. The full length cDNA encoding the rat NPFF1 was digestedwith Sph I (nucleotides 766-1111), and this 345 nucleotide fragment wascloned into the Sph I site of pGEM 3z, containing both sp6 and T7 RNApolymerase promotor sites. The construct was sequenced to confirmsequence identity and orientation. To synthesize antisense strands ofRNA, this construct was linearized with Hind III and T7 RNA polymerasewas used to incorporate radiolabeled nucleotide as described below.

A probe coding for the rat glyceraldehyde 3-phosphate dehydrogenase(GAPDH) gene, a constitutively expressed protein, was used concurrently.GAPDH is expressed at a relatively constant level in most tissue and itsdetection is used to compare expression levels of the rat NPFF1receptors genes in different regions.

Synthesis of probes: NPFF1 and GAPDH cDNA sequences preceded by phagepolymerase promoter sequences were used to synthesize radiolabeledriboprobes. Conditions for the synthesis of riboprobes were: 0.25-1.0 μglinearized DNA plasmid template, 1.5 μl of ATP, GTP, UTP (10 mM each), 3μl dithiothreitol (0.1M), 30 units RNAsin RNAse inhibitor, 0.5-1.0 μl(15-20 units/μl) RNA polymerase, 7.0 μl transcription buffer (PromegaCorp.), and 12.5 μl α³²P-CTP (specific activity 3,000 Ci/mmol). 0.1 mMCTP (0.02-1.0 μl) was added to the reactions, and the volumes wereadjusted to 35 μl with DEPC-treated water. Labeling reactions wereincubated at 37° C. for 60 minutes, after which 3 units of RQ1RNAse-free DNAse (Promega Corp.) were added to digest the template.Riboprobes were separated from unincorporated nucleotides usingMicrospin S-300 columns (Pharmacia Biotech). TCA precipitation andliquid scintillation spectrometry were used to measure the amount oflabel incorporated into the probe. A fraction of all riboprobessynthesized was size-fractionated on 0.25 mm thick 7M urea, 4.5%acrylamide sequencing gels. These gels were apposed to screens and theautoradiograph scanned using a phosphorimager (Molecular Dynamics) toconfirm that the probes synthesized were full-length and not degraded.

Solution hybridization/ribonuclease protection assay (RPA): For solutionhybridization 2.0 μg of mRNA isolated from tissues were used. Negativecontrols consisted of 30 μg transfer RNA (tRNA) or no tissue blanks. AllmRNA samples were placed in 1.5 ml microfuge tubes and vacuum dried.Hybridization buffer (40 μl of 400 mM NaCl, 20 mM Tris, pH 6.4, 2 mMEDTA, in 80% formamide) containing 0.25-2.0 E⁶ counts of each probe wereadded to each tube. Samples were heated at 95° C. for 15 min, afterwhich the temperature was lowered to 55° C. for hybridization.

After hybridization for 14-18 hr, the RNA/probe mixtures were digestedwith RNAse A (Sigma) and RNAse T1 (Life Technologies). A mixture of 2.0μg RNAse A and 1000 units of RNAse T1 in a buffer containing 330 mMNACl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 μl) was added to eachsample and incubated for 90 min at room temperature. After digestionwith RNAses, 20 μl of 10% SDS and 50 μg proteinase K were added to eachtube and incubated at 37° C. for 15 min. Samples were extracted withphenol/chloroform:isoamyl alcohol and precipitated in 2 volumes ofethanol for 1 hr at −70° C. Pellet Paint (Novagen) was added to eachtube (2.0 μg) as a carrier to facilitate precipitation. Followingprecipitation, samples were centrifuged, washed with cold 70% ethanol,and vacuum dried. Samples were dissolved in formamide loading buffer andsize-fractionated on a urea/acrylamide sequencing gel (7.0 M urea, 4.5%acrylamide in Tris-borate-EDTA). Gels were dried and apposed to storagephosphor screens and scanned using a phosphorimager (Molecular Dynamics,Sunnydale, Calif.).

RT-PCR

For the detection of low levels of RNA encoding rat NPFF1, RT-PCR wascarried out on mRNA extracted from rat tissue. Reverse transcription andPCR reactions were carried out in 50 μl volumes using EzrTth DNApolymerase (Perkin Elmer). Primers with the following sequences wereused:

RA Rsnorf2/NPFF F1: CTCCTACTACCAACACTCCTCTCC (SEQ ID NO: 39) RARSNORF2/NPFF1 Bl: ACGGGTTACGAGCATCCAG (SEQ ID NO: 40)

These primers will amplify 490 base pair fragment from nucleotide 574 to1064.

Each reaction contained 0.2 μg mRNA and 0.3 μM of each primer.Concentrations of reagents in each reaction were: 300 μM each of dGTP,DATP, dCTP, dTTP; 2.5 mM Mn(OAc)₂; 50 mM Bicine; 115 mM K acetate, 8%glycerol and 5 units EzrTth DNA polymerase. All reagents for PCR (exceptmRNA and oligonucleotide primers) were obtained from Perkin Elmer.Reactions were carried out under the following conditions: 65° C., 60min; 94° C., 2 min; (94° C., 1 min; 65° C., 1 min) 35 cycles, 72° C., 10min. PCR reactions were size fractionated by agarose gel electrophoresisusing 10% polyacrylamide. DNA was stained with SYBR Green I (MolecularProbes, Eugene, Oreg.) and scanned on a Molecular Dynamics (Sunnyvale,Calif.) Storm 860 in blue fluorescence mode at 450 nM.

Positive controls for PCR reactions consisted of amplification of thetarget sequence from a plasmid construct, as well as reversetranscribing and amplifying a known sequence. Negative controlsconsisted of mRNA blanks as well as primer blanks. To confirm that themRNA was not contaminated with genomic RNA, samples were digested withRNAses before reverse transcription. Integrity of RNA was assessed byamplification of mRNA coding for GAPDH.

Localization of mRNA Coding for NPFF-like Receptors (hNPFF2) UsingRT-PCR

For the detection of low levels of RNA encoding hNPFF2 RT-PCR wascarried out on mRNA extracted from tissue. Reverse transcription and PCRreactions were carried out in 50 μl volumes using EzrTh DNA polymerase(Perkin Elmer). Primers with the following sequences were used:

(SEQ ID NO: 41) JB 249: 5′-GATCAGTGGATTGGTCCAGGGAATATC-3′ (SEQ ID NO:42) JB 250: 5′-CCAGGTAGATGTTGGCAAACAGCAC-3′

These primers will amplify a 332 base pair fragment from TMIII to TMV.

Each reaction contained 0.1 ug mRNA and 0.3 uM of each primer.Concentrations of reagents in each reaction were 300 uM each of dGTP,DATP, dCTP, dTTP, 2.5 mM Mn(OAc)2, 50 mM Bicine, 115 mM potassiumacetate, 8% glycerol and 5 units EzrTth DNA polymerase. All reagents forPCR (except mRNA and oligonucleotide primers) were obtained from PerkinElmer. Reactions were carried out under the following conditions: 65° C.60 min., 94° C. 2 min, (94° C., 1 min, 65° C. 1 min) 35 cycles, 72° C.10 min. PCR reactions were size fractionated by gel electrophoresisusing 10% polyacrylamide. DNA was stained with SYBR Green I (MolecularProbes, Eugene Oreg.) and scanned on a Molecular Dynamics (Sunnyvale,Calif.) Strom 860 in blue fluorescence mode at 450 nm.

Positive controls for PCR reactions consisted of amplification of thetarget sequence from a plasmid construct, as well as reversetranscribing and amplifying a known sequence. Negative controlsconsisted of mRNA blanks and primer blanks. To confirm that the mRNA wasnot contaminated with genomic DNA, samples were digested with RNAsesbefore reverse transcription. Integrity of RNA was assessed byamplification of mRNA coding for GAPDH.

Localization of mRNA Coding for Human and Rat NPFF Receptors

Materials and Methods

Quantitative RT-PCR using a fluorogenic probe with real time detection:Quantitative RT-PCR using fluorogenic probes and a panel of mRNAextracted from human and rat tissue was used to characterize thelocalization of NPFF rat and human RNA. This assay utilizes twooligonucleotides for conventional PCR amplification and a third specificoligonucleotide probe that is labeled with a reporter at the 5′ end anda quencher at the 3′ end of the oligonucleotide. In the instantinvention, FAM (6-carboxyfluorescein) and JOE (6carboxy-4.5-dichloro-2,7-dimethoxyfluorescein) were the two reportersthat were utilized and TAMRA (6-carboxy-4,7,2,7′-tetramethylrhodamine)was the quencher. As amplification progresses, the labeledoligonucleotide probe hybridizes to the gene sequence between the twooligonucleotides used for amplification. The nuclease activity of Taq,or rTth thermostable DNA polymerases is utilized to cleave the labeledprobe. This separates the quencher from the reporter and generates afluorescent signal that is directly proportional to the amount ofamplicon generated. This labeled probe confers a high degree ofspecificity. Non-specific amplification is not detected as the labeledprobe does not hybridize. All experiments were conducted in a PE7700Sequence Detection System (Perkin Elmer, Foster City Calif.).

Quantitative RT-PCR: For the detection of RNA encoding NPFF receptors,quantitative RT-PCR was performed on mRNA extracted from tissue. Reversetranscription and PCR reactions were carried out in 50 μl volumes usingrTth DNA polymerase (Perkin Elmer). Primers with the following sequenceswere used:

NPFF1 Human:

(SEQ ID NO: 60) Forward primer: NPFF1h-913F 5′-CTGGTCACCGTCTACGCCTT-3′(SEQ ID NO: 61) Reverse primer: NPFF1h-1016R 5′-CCGCGGCGGAAGTTCT-3′ (SEQID NO: 62) Fluorogenic oligonucleotide probe: NPFF1h-962T5′(6-FAM)-ACAGCAGCGCCAACCCCATCAT-(TAMRA)3′

NPFF2 Human:

(SEQ ID NO: 63) Forward primer: NPFF2h-828F 5′-CCTGATTGTGGCCCTGCT-3′(SEQ ID NO: 64) Reverse primer: NPFF2h-916R5′-CATTTGGAGAAAGGTCAGCGTAG-3′ (SEQ ID NO: 65) Fluorogenicoligonucleotide probe: NPFF2h-855T5′(6-FAM)-CTCATGGCTGCCCCTGTGGACTCAAT-(TAMRA)3′

NPFF1 Rat

(SEQ ID NO: 66) Forward primer: NPFF1r-412F 5′-GCTGTGGAAAGGTTCCGCT-3′(SEQ ID NO: 67) Reverse primer: NPFF1r-474R 5′-CGCCTTCCGAAGGGTCA-3′ (SEQID NO: 68) Fluorogenic oligonucleotide probe: NPFF1r-433T5′(6-FAM)-ATCGTGCACCCTTTCCGCGAGAA-(TAMRA)3′

NPFF2 Rat

Forward primer: NPFF2r deg-690F (SEQ ID NO: 69)5′-GAGGATCTACACCACCGTGCTATT-3′ Reverse primer: NPFF2r deg-776R (SEQ IDNO: 70) 5′-GAAGCCCCAATCCTTGCATAC-3′ Fluorogenic oligonucleotide probe:NPFF2r-722T (SEQ ID NO: 71)5′(6-FAM)-TCTACCTGGCTCCACTCTCCCTCATTGTT-(TAMRA)3′

Using these primer pairs, amplicon length is 103 bp for human NPFF1, 88bp for human NPFF2, 62 bp for rat NPFF1, and 86 bp for rat NPFF2. Eachhuman RT-PCR reaction contained 50 ng mRNA and each rat RT-PCR reactioncontained 100 ng total RNA. Oligonuceotide concentrations were: 500 nMof forward and reverse primers, and 200 nM of fluorogenic probe.Concentrations of reagents in each reaction were: 300 μM each of dGTP;DATP; dCTP; 600 μM UTP; 3.0 mM Mn(OAc)2; 50 mM Bicine; 115 mM potassiumacetate, 8% glycerol, 5 units rTth DNA polymerase, and 0.5 units ofuracil N-glycosylase. Buffer for RT-PCR reactions also contained a fluorused as a passive reference (ROX: Perkin Elmer proprietary passivereference I). All reagents for RT-PCR (except mRNA and oligonucleotideprimers) were obtained from Perkin Elmer (Foster City, Calif.).Reactions were carried using the following thermal cycler profile: 50°C. 2 min., 60° C. 30 min., 95° C. 5 min., followed by 40 cycles of: 94°C., 20 sec., 62° C. 1 min.

Positive controls for PCR reactions consisted of amplification of thetarget sequence from a plasmid construct when available. Standard curvesfor quantitation of human and rat NPFF1 and NPFF2 were constructed usingRNA extracted from whole brain. Negative controls consisted of mRNAblanks, as well as primer and mRNA blanks. To confirm that the mRNA wasnot contaminated with genomic DNA, PCR reactions were carried outwithout reverse transcription using Taq DNA polymerase. Integrity of RNAwas assessed by amplification of RNA coding for cyclophilin orglyceraldehyde 3-phosphate dehydrogenase (GAPDH). Following reversetranscription and PCR amplification, data was analyzed using PerkinElmer sequence detection software. The fluorescent signal from each wellwas normalized using an internal passive reference, and data was fitteda standard curve to obtain relative quantities of NPFF mRNA expression.

Receptor Autoradiographic Experiments Localizing NPFF Receptor Subtypesin the Rat CNS

Animals

Male Sprague-Dawley rats (Charles Rivers, Rochester, N.Y.) wereeuthanized using CO₂, decapitated, and their brains and peripheraltissues were immediately removed and rapidly frozen on crushed dry ice.Coronal sections of brain tissues were cut at 20 μm using a cryostat,thaw-mounted onto gelatin-coated slides then stored at −20° C. untilbinding assay.

Materials

[¹²⁵][D-Tyr¹-(NMe)Phe³]NPFF (specific activity 2200 Ci/mmol wassynthesized by iodination with chloramine-T from NEN (Boston, Mass.).BIBP 3226 was from RBI (Natick, M A). Frog pancreatic polypeptide (frogPP)(Rana Temporaria) was from Peninsula (Belmont, Calif.), andNeuropeptide FF (NPFF) was from (Bachem, King of Prussia, Pa.).

In Vitro Autoradiography

Tissue sections were allowed to equilibrate to room temperature for onehour. Sections were then incubated at 25° C. for 2 hours in 50 mMTris-HCl buffer, pH 7.4, containing 1 mM NaCl, 1 mM MgCl₂₁ 0.1% BovineSerum Albumin (Boehringer Mannheim, Indianapolis, Ind.) and 0.05 nM[¹²⁵I] [D-Tyr¹-(NMe)Phe³]NPFF. Adjacent sections were incubated in thepresence of 300 nM BIBP 3226 to selectively displace binding to NPFF1 or300 nM frog PP to selectively displace binding to NPFF2. Nonspecificbinding was determined by including 1 μM unlabeled NPFF in theincubation buffer. Sections were then washed four times, 5 minutes each,in 4° C. 50 mM Tris-buffer pH 7.4 then rapidly dipped in ice-colddistilled water to remove salts. Tissues were then dried under a streamof cold air. The sections were subsequently apposed together with¹²⁵I-plastic standard scales, to Kodak BIOMAX MS Scientific Imaging Film(Eastman Kodak Company, Rochester, N.Y.) for three days at roomtemperature. Films were developed using a Kodak M35A X-OMAT Processor(Eastman Kodak Company, Rochester, N.Y.). Specific [¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF binding to NPFF1 and NPFF2 receptors wasinterpreted by observation of the remaining optical density on theautoradiogram in the various regions of rat brain in the presence of theappropriate displacers.

In Vivo Experiments

Effects of NPFF on Blood Pressure in Normotensive Rats

It has been demonstrated that the intravenous administration of NPFFproduces an increase in mean arterial blood pressure (MAP) (71). Thefollowing experiments were designed to determine which subtype of NPFFreceptor mediates this effect.

Experimental Methods

Rats were anesthetized with urethane and PE50 cannulae were placed inthe femoral artery and vein for blood pressure monitoring and drugadministration, respectively. After stabilization, rats wereadministered 200 μl saline vehicle; the agonists NPFF, frog pancreaticpolypeptide (fPP), or the NPY-Y₁-selective agonist pLeu, Pro-NPY[(Leu³¹,Pro³⁴)-neuropeptide Y (porcine)]; or the NPY-Y₁ receptorantagonist BIBP 3226.

Effects of NPFF on the Micturition Reflex in Anesthetized Rats

The effects of compounds on the micturition reflex were assessed in the“distension-induced rhythmic contraction” (DIRC) model in rats. Thismodel of the micturition reflex has been described in previouspublications (e.g. 72, 73).

DIRC Model

Female Sprague Dawley rats weighing approximately 300 g wereanesthetized with subcutaneous urethane (1.2 g/kg). The trachea wascannulated with PE240 tubing to provide a clear airway throughout theexperiment. A midline abdominal incision was made and the left and rightureters were isolated. The ureters were ligated distally (to preventescape of fluids from the bladder) and cannulated proximally with PE10tubing. The incision was closed using 4-0 silk sutures, leaving the PE10lines routed to the exterior for the elimination of urine. The bladderwas canulated via the transurethral route using PE50 tubing inserted 2.5cm beyond the urethral opening. This cannula was secured to the tailusing tape and connected to a pressure transducer. To prevent leakagefrom the bladder, the cannula was tied tightly to the exterior urethralopening using 4-0 silk.

To initiate the micturition reflex, the bladder was first emptied byapplying pressure to the lower abdomen, and then filled with normalsaline in 100 μl increments (maximum=2 ml) until spontaneous bladdercontractions occurred (typically 20-40 mmHg at a rate of one contractionevery 2 to 3 minutes. Once a regular rhythm was established, vehicle(saline) or test compounds were administered intravenously to examinetheir effects on bladder activity. The effect of a compound whichinhibited the micturition reflex was expressed as its “disappearancetime”, defined as the time between successive bladder contractions inthe presence of the test compound minus the time between contractionsbefore compound administration.

Chromosomal Localization of Human NPFF1 and NPFF2 Receptor Genes.

Chromosomal localization for human NPFF1 and NPFF2 receptor genes wasestablished using a panel of radiation hybrids prepared by the StanfordHuman Genome Center (SHGC) and distributed by Research Genetics, Inc.The “Stanford G3” panel of 83 radiation hybrids was analyzed by PCRusing the same primers, probes and thermal cycler profiles as used forlocalization. 20 ng of DNA was used in each PCR reaction. Data wassubmitted to the RH Server (SHGC) which linked the NPFF1 and NPFF2 genesequences to specific markers. NCBI LocusLink and NCBI GeneMap '99 wereused for further analysis of gene localization.

Results and Discussion

Cloning and Sequencing

rNPFF1 and hNPFF1

100 ng genomic DNA was subjected to MOPAC PCR with two degenerateprimers designed based on the sixth and seventh transmembrane domains ofover 180 receptors from the rhodopsin superfamily of G protein-coupledreceptors. Two products from this reaction, MPR3-RGEN-31 andMPR3-RGEN-45 were found to be identical clones of a novel DNA sequencenot found in the Genbank databases (Genembl, STS, EST, GSS), which had30-40% amino acid identity with the known receptors dopamine D2, orexin1, GALR1, angiotensin 1B and 5HT-2b. This novel clone was given the nameSNORF2.

The full-length SNORF2 sequence was acquired by screening rathypothalamic cDNA libraries by PCR using specific SNORF2 oligonucleotideprimers. Pools of the rat hypothalamic cDNA library “I” were screened byPCR with SNORF2-specific primers JAB208 and JAB209. This screen yieldeda positive pool I36. Successive PCR screening of sub-pools of this poolfollowed by high stringency hybridization of isolated colonies from thepositive sub-pool I36-17 with the SNORF2-specific oligonucleotide probeindicated that the isolated clone I36E-17-1B-1 contained at least apartial clone of SNORF2. Sequencing of I36E-17-1B-1 revealed that thisinsert contained the coding region from the TMIII-TMIV loop through thestop codon, including some 3′ untranslated sequence. From this sequence,a new forward primer, JAB221, was designed in TMV. PCR screening of asecond rat hypothalamic cDNA library “J” with primers JAB221 and JAB209,and subsequent colony hybridization with the JAB211 probe on a lowcomplexity positive sub-pool resulted in the isolation of a SNORF2 cloneJ-13-16-A1. This clone contained the full-length coding sequence ofSNORF2 (1296 bp) with approximately 200 bp 5'untranslated sequence and1.3 kb 3′ untranslated sequence. The nucleotide sequence of SNORF2 andits translated amino acid sequence are represented in FIG. 1 (SEQ IDNO:1) and FIG. 2 (SEQ ID NO:2), respectively. As shown in FIG. 1, SNORF2contains two potential initiating methionines upstream of TMI.

Hydophobicity (Kyte-Doolittle) analysis of the amino acid sequence ofthe full-length clone indicates the presence of seven hydrophobicregions, which is consistent with the seven transmembrane domains of a Gprotein-coupled receptor. The seven expected transmembrane domains aremapped out in FIG. 2. A comparison of nucleotide and peptide sequencesof SNORF2 with sequences contained in the Genbank/EMBL/SwissProtPlusdatabases reveals that the amino acid sequence of this clone is mostrelated to the orexin 1 and 2 receptors (45% and 40% identity,respectively) as well as the neuropeptide Y receptors Y1, Y2 and Y4(˜30% identity). Further homology analysis of SNORF2 against theSynaptic Pharmaceutical Corporation in-house database revealed thatSNORF2 has a very high degree of identity with a proprietary SynapticPharmaceutical Corporation human partial GPCR clone named PLC29b (85%nucleotide identity, 93% amino acid identity). PLC29b was originallyisolated from a human genomic library using oligonucleotide probes forNPY4, and includes part of the amino terminus and TMs I to IV. Partialnucleotide and amino acid sequence of PLC29b (human SNORF2) isrepresented in SEQ ID NO:3 and SEQ ID NO:4, respectively. Based onsequence similarity, PLC29b appears to be a partial clone of the humanhomologue of SNORF2. Therefore, this human homolog of SNORF2 has beennamed hNPFF1. A GAP alignment demonstrating the high homology betweenthese species homologues is represented in FIG. 3.

SNORF2 has several potential protein kinase C (PKC) phosphorylationmotifs throughout its amino acid sequence: threonine 154 in the secondintracellular loop, threonine 263 and serine 264 in the thirdintracellular loop, and serine 363 in the intracellular carboxy-terminaltail. It also has four potential N-linked glycosylation sites atasparagines 10 and 18 in the amino-terminal tail and at asparagines 113and 195 in the first and second extracellular loops, respectively.

hNPFF2

In analyzing the sequence of rNPFF1 and its homology to other sequencesin GenBank, a 532 bp EST with the accession number AA449919 wasidentified which had a high degree of identity to rNPFF1. Thetranslation of this sequence indicated that it coded for the regionbetween the first extracellular loop and the beginning of the sixthtransmembrane domain of a G protein-coupled receptor (GPCR). AlthoughAA449919 was documented as being similar to the Drosophila melanogasterNPY receptor (accession number P25931), it was found that the amino acidsequence encoded by this EST was much more similar to NPFF1. Thepredicted amino acid sequence of AA449919 and rNPFF1 are 50% identical,while the amino acid sequence of the Drosophila NPY receptor is only 31%identical to the translation of AA449919. Because of the high degree ofidentity between AA449919 and rNPFF1, AA449919 was given the namehNPFF2, representing a member of a novel family of NPFF receptors ofwhich there is currently only one member, NPFF1.

The full length sequence of NPFF-like (hNPFF2) was acquired by 5′/3′RACE using human spleen cDNA as a template, as described above,demonstrating that the coding region of hNPFF-like (hNPFF2) is 1260 bp,coding for a protein of 420 amino acids. Sequencing of clones fromseveral independent PCR reactions using spleen, heart, and spinal cordcDNA as templates and subsequent alignment of these clones withSequencer 3.0 was used to confirm the sequence of hNPFF-like (hNPFF2).The full-length nucleotide sequence of human NPFF2 is shown in FIG. 4(SEQ ID NO:5), and its translated amino acid sequence is shown in SEQ IDNO:6. The seven putative transmembrane domains of hNPFF-like (hNPFF2)are defined in FIG. 5.

Like the original EST AA449919, the amino acid sequence encoded by thefull-length DNA sequence of hNPFF2 is most similar to rNPFF1 (48%identity), as shown in the GAP alignment between the two receptors inFIG. 6. The next-best matches in SWPLUS to full-length hNPFF2 are theDrosophila NPYR (accession number P25931, 34% identity) and TLR2(accession number P30975, 32% identity), human orexin 1 and 2 receptors(O43613, 31% and O43614, 29%, respectively) and human NPY1 and Y4receptors (P25929, 31% and P50391, 32%, respectively). A Blast search ofthe EST database using the full-length nucleotide sequence of hNPFF2revealed an EST (Accession number AA449920) that is identical to hNPFF2from the end of TM7 through the stop codon. ESTs AA44919 and AA44920 arethe same clone sequenced from 5′ end or the 3′ end, respectively.

hNPFF2 contains several potential N-linked glycosylation sites. Thefirst three sites, asparagines 8, 20, and 31 are in the N-terminalextracellular domain. Another potential N-linked glycosylation site, atposition 198, is in the second extracellular loop. This receptor alsocontains one potential PKC phosphorylation site at threonine 156 in thesecond intracellular loop, and two potential PKC phosphorylation sitesin the third intracellular loop at threonine 254 and serine 266.

hNPFF1

The sequence of hNPFF1 from the initiating methionine to TMIV wasdetermined to be present in a partial clone, plc29b, found in a SynapticPharmaceutical Corporation in-house database. Additional sequence,including TMIV through the stop codon, was determined by sequencing avector-anchored PCR product from a human cosmid library clone identifiedby hybridization with a ³²P-labeled probe (BB609) corresponding to theII/III loop of plc29b. Next, a human spinal cord library was screened byPCR using primers designed against the partial hNPFF1 sequence, BB729and BB728. One positive pool, W4, was subdivided and a positive sub-poolwas screened by colony hybridization with a ³²P-labeled probe from TMIIof hNPFF1, BB676. Plasmid DNA was isolated for clone W4-18-4, renamedBO98, and DNA sequencing revealed that it was full-length but in thewrong orientation for expression in the expression vector pEXJ. Toobtain a full-length hNPFF1 construct in the correct orientation, BO98was amplified with BB757 and BB758, and the resulting product ligatedinto pcDNA3.1 and transformed into DH5α cells. The sequence of one ofthese transformants was identical to the hNPFF1 sequence previouslydetermined from the consensus of BO98 and the two cosmid clones. Thishuman NPFF1 construct in pcDNA3.1 in the correct orientation was renamedBO102.

The hNPFF1 clone contains an open reading frame with 1293 nucleotides(see FIG. 7 and SEQ ID NO:7) and predicts a protein of 430 amino acids(see FIG. 8 and SEQ ID NO:8). Seven transmembrane domains predicted byhydrophobicity analysis are indicated in FIG. 8. The sequence of hNPFF1was determined to be most similar to the rat NPFF1 (86% nucleotideidentity, 87% amino acid identity) and human NPFF2 (56% nucleotideidentity, 49% amino acid identity (FIG. 9)). The human NPFF1 receptoralso shares homology with human orexin₁ (53% nucleotide identity, 35%amino acid identity), human orexin₂ (43% nucleotide identity, 33% aminoacid identity), human NPY₂ (47% nucleotide identity, 31% amino acididentity), human CCK_(A) (46% nucleotide identity, 32% amino acididentity), and human CCK_(B) (46% nucleotide identity, 26% amino acididentity).

Isolation of the Rat NPFF2 Homologue

A fragment of the rat homologue of NPFF2, from TMIV to TMVI, wasamplified from rat genomic DNA, rat hypothalamic cDNA and rat spinalcord cDNA by reduced stringency PCR using oligonucleotide primersdesigned against the human NPFF2. Additional sequence was obtained byamplifying rat spinal cord cDNA under reduced stringency using PCRprimers designed against the rat NPFF2 fragment along with primerscorresponding to the NH₂— and COOH-termini of the human NPFF2 receptor.This resulted in the identification of a rat NPFF2 fragment from TMI toTMVII.

The remaining sequence of the rat NPFF2 receptor was acquired byscreening a rat genomic phage library with an oligonucleotide probecorresponding to the second extracellular loop and TMV of rat NPFF2.Southern blot analysis of three isolated plaques with this same probeidentified a 3.5 kb fragment which was subcloned and sequenced,revealing the COOH terminus and some 3′UT. A subsequent Southern blotanalysis using an oligonucleotide probe corresponding to TMI of ratNPFF2 identified a 2.1 kb fragment which was subcloned and sequenced,revealing the NH₂ terminus and some 5′UT.

The full-length rat NPFF2 clone was amplified from rat spinal cord cDNAusing a sense PCR primer corresponding to the 5′UT and an antisenseprimer corresponding to the 3′UT, and subcloned into pcDNA3.1.Sequencing of 5 independent PCR products revealed an open reading frameof 1251 bp (SEQ ID NO:43) that is predicted to encode a protein of 417amino acids (SEQ ID NO:44) (see also FIGS. 17A-17C and 18A-18B). Inaddition, several potential allelic variations were identified andverified by sequencing additional genomic DNA PCR products. The allelicvariations are at the following nucleotide positions (relative to FIGS.17A-17C (SEQ ID NO:43)): position 913 can be either G or A, position 949can be either C or T, position 955 can be either C or T, and position1151 can be either C or T. None of these variants alter the predictedamino acid sequence. One construct, whose nucleotide sequence is shownin FIGS. 17A-17C (SEQ ID NO:43) was renamed pcDNA3.1-rNPFF2-f.

Hydophobicity (Kyte-Doolittle) analysis of the amino acid sequence ofthe full-length clone indicates the presence of seven hydrophobicregions, which is consistent with the seven transmembrane domains of a Gprotein-coupled receptor. The seven expected transmembrane domains areindicated in FIGS. 18A-18B. A comparison of nucleotide and peptidesequences of rat rNPFF2 with sequences contained in the Genbank, EMBLand SwissProtPlus databases reveals that the nucleotide sequence of thisclone is 81% identical to an orphan GPCR NPGPR (GenBank accession numberAF119815), and the amino acid sequence of this clone is most related toorexin-1 and orexin-2 (34% amino acid identities), NPY2 (32% amino acididentity) and GIR (31% amino acid identity). There were no sequences inthe Genbank databases (Genembl, STS, EST, GSS, or SwissProt) that wereidentical to rat NPFF2. The rat and human NPFF2 receptors share 81%nucleotide and 78% amino acid identities (FIGS. 19A-19B). The rat NPFF2and rat NPFF1 receptors share 55% nucleotide and 50% amino acididentities (FIGS. 20A-20B).

Rat NPFF2 has five potential N-linked glycosylation sites, atasparagines 8, 20 and 31 in the amino-terminal tail, at asparagine 198in the second extracellular loop and at asparagine 324 in the seventhtransmembrane domain. It also has three potential protein kinase C (PKC)phosphorylation motifs at threonine 156 in the second intracellularloop, and at threonine 254 and serine 265 in the third intracellularloop. NPFF2 also has two potential casein kinase II phosphorylationsites at threonine 102 in the second transmembrane domain and at serine403 in the carboxy-terminal tail.

Electrophysiology

NPFF1

Oocytes injected with both SNORF2 and chimeric Gα_(q/z) synthetic RNAsgenerated robust inward currents in response to NPFF and the relatedpeptide A-18-F-amide at 1 μM (FIGS. 10A, 10B). Control oocytes receivingonly G-protein synthetic RNA were unresponsive to these peptides.Responses to NPFF were concentration-dependent with a threshold foractivation of inward current at 30 nM. The C-terminal tetrapeptidePQRF-amide also elicited responses at a concentration of 10 μM (FIG.10C). Analogs of NPFF containing a tyrosine residue at the N-terminus orinternally including Y-8-F-amide, [tyr⁹]A-18-F-amide and Y-18-F-amidealso displayed activity at 1 μM. Unrelated neuropeptides and otherneurotransmitters including melanin concentrating hormone, orexin B,PYY, 5-HT, nociceptin, galanin and CCK failed to activate oocytesinjected with the SNORF2 synthetic RNA. The functional responsiveness toNPFF and related peptides strongly suggests that SNORF2 encodes areceptor for neuropeptide FF (NPFF); therefore SNORF2 was renamed NPFF1.Similarly, SNORF2-like was renamed NPFF-like.

Oocytes injected with NPFF1 and not the chimeric G-protein synthetic RNAfailed to generated responses to NPFF. This observation supports thehypothesis that NPFF1 couples to G-proteins of the Gα_(i)/Gα_(o)/Gα_(z)class, and by virtue of the N-terminal portion of Gα_(q/z), subsequentlyactivates phospholipase C. In oocytes expressing both NPFF1 andGα_(q/z), Cl⁻ currents were abolished by prior injection of 10 mM EGTA,demonstrating the Ca⁺⁺ dependence of these currents.

NPFF2

Oocytes injected with both the NPFF-like PCR product and chimericGα_(q/z) synthetic RNAs generated large inward currents in response to 1μM NPFF (FIG. 11A). A-18-F-amide and PQRF-amide also at 1 μM activatedsimilar inward currents, although the magnitude of currents generated byPGRF-amide were smaller. No activity was observed using FMRF-amide at 1μM. The unrelated neuropeptides orexin A, NPY, galanin, and neurokinin Aat 1 μM also failed to activate responses in oocytes injected withNPFF-like mRNA (FIG. 11B). Oocytes injected with both the NPFF-likeplasmid (BO89) and chimeric Gα_(q/z) synthetic RNAs also produced robustcurrents in response to NPFF (FIG. 11C). Based on these results,NPFF-like was renamed NPFF2. Oocytes injected with NPFF2 and notchimeric G-protein mRNA failed to generate responses to NPFF. Thisobservation supports the hypothesis that NPFF2 couples to G-proteins ofthe Gα_(i)/Gα_(o)/Gα_(z) class, and by virtue of the N-terminal portionof Gα_(q/z), subsequently activates phospholipase C.

Microphysiometry

CHO cells transiently expressing either NPFF1 alone or NPFF1 accompaniedby the chimeric protein Gq/Gz produced robust increases in metabolismwhen exposed to either. NPFF or the related peptide A-18-F-amide asevidenced by increased rates of extracellular acidification whenmeasured by the microphysiometric technique (FIGS. 12A and 12B). Whereascontrol cells, not expressing NPFF1, produced no increase inacidification rates to either NPFF or A-18-F-amide. In all cases theNPFF1 mediated responses were dose-dependent. CHO cells transfected withNPFF1 alone produced an EC50 value of 19.3 nM for NPFF while cellstransfected NPFF1 and the chimeric Gz/Gq produced an EC50 of 27.7 nM forNPFF. Challenges with A-18-F-amide were conducted only on cells that hadbeen transfected with NPFF1 alone. These cells produced an EC50 value of150 nM for A-18-F-amide. The functional responsiveness to NPFF andA-18-F-amide supports the notion that NPFF1 encodes a receptor forneuropeptide FF (NPFF).

Radioligand Binding Assays

Cos-7 cells transiently expressing the gene encoding the novel rat NPFF1receptor were used for pharmacological evaluation. Membranes harvestedfrom transiently transfected Cos-7 cells exhibited high affinity,saturable [¹²⁵I]D-Tyr-NPFF ([D-Tyr¹(NMe)Phe³]NPFF) binding. Nonlinearanalysis of [¹²⁵I]D-Tyr-NPFF saturation data yielded an equilibriumdissociation constant (K_(d)) of 0.335±0.045 nM and a binding sitedensity (B_(max)) of 180±11 fmol/mg protein. Specific [¹²⁵I]D-Tyr-NPFFbinding was about 50% of total binding at a radioligand concentrationequal to the K_(d) value. Mock-transfected host cells did not displayspecific [¹²⁵I]D-Tyr-NPFF binding.

To further assess the pharmacological identity of the newly isolatedNPFF1 receptor gene, detailed binding properties of cloned NPFF1receptor were determined from nonlinear analysis of competition of highaffinity [¹²⁵I]D-Tyr-NPFF binding. The rank order of affinity ofcompounds to displace specific [¹²⁵I]D-Tyr-NPFF binding is shown inTable 1.

The binding profile of rat NPFF1 was compared to that of rat spinal cordmembranes. Interestingly some differences were observed in thepharmacological profile between the two preparations. (See * Table 2).Notably, fPP did not displace the binding on the NPFF1 receptor up to 1uM whereas it displayed a high affinity at the rat spinal cord.Furthermore, several compounds displayed significantly differentaffinities between NPFF1 receptor and the spinal cord membranes. Thesecompounds are highlighted in Table 1 and are ([¹²⁵I]D-Tyr-NPFF,A18Famide, Y8Famide, [Y⁹]A18Famide, Dynorphin A 1-13, Neuropeptide F andMet-Enk-NH2. These data indicate the presence of additional NPFFreceptor subtypes on the rat spinal cord.

Additional pharmacological evaluation was done using 293 human embryonickidney cells (HEK-293 cells) transiently expressing the genes encodingthe human NPFF1, NPFF2, and rat NPFF1 receptors, as well as Cos-7 cellsexpressing the rat NPFF2 receptors. Nonlinear analysis of[¹²⁵I]D-Tyr-NPFF saturation binding data yielded equilibriumdissociation constants (K_(d)) of 0.46±0.10 and 0.17±0.04 nM for thehuman NPFF1 and NPFF2, and of 0.65±0.22 and 0.17±0.02 nM for the ratNPFF1 and NPFF2 receptors, respectively. The binding affinities (pKi) ofvarious NPFF-related peptides were derived from competition bindingassay using [¹²⁵I]D-Tyr-NPFF as a ligand. In agreement with the datashown in Table 1, fPP showed 31- and 77-fold greater affinity for therat and human NPFF2 receptors, respectively, when compared to the NPFF1receptors (see Table 3). The other peptides studied showed overallsimilar binding affinities for both the rat and human NPFF1 and NPFF2receptors. NPFF receptors displayed high affinity for FMRF amide andlower binding affinity for the D-Met analog, suggesting the existence ofstereoselectivity for this peptide.

The ability of NPFF1 receptors to functionally couple to PI was testedusing intact Cos-7 cells transiently expressing NPFF1. Fulldose-response curves were determined for NPFF-mediated total IP release(FIG. 13A). NPFF stimulated total IP release with an EC50 of 23 nM andan Emax of approximately 200% basal. This weak stimulation was mostprobably mediated by NPFF1 coupling to a Gi/Go G-protein via βγ-inducedPI turnover, since the response was abolished by pretreatment withpertussis toxin but not cholera toxin. In contrast, a robust stimulationof total IP release was observed following NPFF in Cos-7 cellstransfected with both the NPFF1 receptor and the Gq/Gz chimera (FIG.13B). NPFF stimulated total IP release with an EC50 of 2.95 nM, and anEmax of approximately 1500% basal. As anticipated, neither PTX nor CTXattenuated this response. Similar to what was observed in oocytes, thissuggests a coupling in Cos-7 cells to G-proteins of theGα_(i)/Gα_(o)/Gα_(z) class.

The coupling of human NPFF1 and NPFF2 receptors to the activation ofintracellular second messenger pathways was studied further in COS-7cells co-transfected with the Gq/Gz chimera. In such cells, NPFFelicited an increase in intracellular calcium when either the humanNPFF1 or NPFF2 were transfected, and no response was observed in cellsthat were only transfected with the Gq/Gz chimera. As shown in Table 4,PQRF amide was a full agonist in cells expressing either the NPFF1 orNPFF2 receptors. However, only cells expressing the human NPFF2responded with an intracellular calcium response to fPP, no response wasobserved in cells expressing the human NPFF1 receptor, suggesting thatfPP is an NPFF2-selective agonist.

TABLE 1 pKi for cloned rat NPFF1 receptor binding in COS-7 cellsCOMPOUND MEAN SEM n NPFF(F-8-Fa) 8.535 0.02 2 (D-Tyr¹-(NMe)Phe³)NPFF8.549 0.13 4 A18Fa 7.495 0.11 2 PQRFa 8.182 0.03 2 FMRFa 8.481 0.05 2YFa 8.382 0.22 2 [Y⁹]A18Fa 7.558 0.12 2 hPP 5 0 2 fPP 5.5 0.35 2substance P 5 0 2 Dynorphin A1-13 6.838 0.29 2 (3D)Y8Fa 8.623 0.44 4(2D)Y8Fa 8.33 0.15 4 CCK8 5 0 2 galanin 5 0 2 dopamine 5 0 2 naloxone 50 2 CGRP 5 0 2 AF-1 6.634 0.13 2 AF-2 7.023 0.41 2 SchistFLRF 5.96 0.682 Met5-Arg6-Phe7-Enk- 7.35 0.22 4 NH2 Met5-Arg6-Phe7-Enk-OH 5 0 2Neuropeptide F 6.11 0.06 4 desamino-nor-Y8Ra 7.27 0.1 3 (2DME)Y8Fa 9.20.01 3 L-arginine 5 0 1 D-arginine 5 0 1 desipramine 5 0 1 fenfluramine5 0 1 harmine 5 0 1 levocabastine 5 0 1 ibogaine 5 0 1 ritanserine 5 0 1a-MSH 5 0 1 Tyr-MIF-1 5 0 1 nociceptin 5 0 1 nocistatin 5 0 1 PMRFa 8.550.06 2 FTRF 7.87 0.1 2 FFRF 8 0 2

TABLE 2 pKi for rat spinal cord membrane receptor binding COMPOUND MEANSEM n NPFF(F-8-fa) 9.055 0.08 2 (D-Tyr¹-(NMe)Phe³)NPFF *9.724 0.25 4A18Fa *9.000 0.21 2 PQRFa 8.541 0.07 2 FMRFa 8.493 0.23 2 Y8Fa *9.1890.06 2 [Y⁹]A18Fa *8.502 0.01 2 hPP 5 0 3 fPP *9.118 0.06 3 substance P 50 1 Dynorphin A1-13 *5.700 0.5 2 (3D)YBFa 9.123 0.12 4 (2D)Y8Fa *9.2120.23 4 CCK8 5 0 2 galanin 5 0 2 dopamine 5 0 2 naloxone 5 0 2 CGRP 5 0 2AF-1 *7.563 0.47 2 AF-2 *7.965 0.24 2 SchistFLRF 6.39 0.23 2 Met-Enk-NH2*8.400 0.08 4 Met-Enk-OH 5 0 2 Neuropeptide F *8.100 0.1 3desamino-nor-Y8Ra 7.51 0.07 3 (2DME)Y8Fa 9.57 0.3 4 L-arginine 5 0 1D-arginine 5 0 1 desipiramine 5 0 1 fenfluramine 5 0 1 harmine 5 0 1levocabastine 5 0 1 ibogaine 5 0 1 ritanserine 5 0 1 α-MSH 5 0 1Tyr-MIF-1 5 0 1 nociceptin 5 0 1 nocistatin 5 0 1 PMRFa 9.37 0.11 2 FTRF8.16 0.16 2 FFRF 8.98 0.001 2 AF-1 = FMRF-like peptideH₂N-Lys-Asn-Gln-Phe-Ile-Arg-Phe-NH₂ AF-2H-Lys-His-Gln-Tyr-Leu-Arg-Phe-NH₂ Schisto(FLRFNH₂) =Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-amide Met⁵, Arg⁶, Phe⁷- NH₂ =enhephalinamide Met⁵, Arg⁶, Phe⁷- OH = enhephalin

TABLE 3 pKi of NPFF-related peptides at cloned human and rat NPFFreceptors in 293 human embryonic kidney cells (HEK-293 cells) human ratNPFF1 NPFF2 NPFF1 NPFF2 pKi ± SEM (D-Tyr¹- 8.1 ± 0.06 8.5 ± 0.08 8.8 ±0.005 8.7 ± 0.16 (NMe)Phe³) NPFF fPP 5.9 ± 0.09 7.4 ± 0.13 5.4 ± 0.107.3 ± 0.02 FMRF amide 9.1 ± 0.19 8.4 ± 0.02 8.7 ± 0.01 8.0 ± 0.02D-Met-FMRF amide 6.6 ± 0.24 6.4 ± 0.03 6.2 ± 0.09 6.2 ± 0.03 A18Fa 7.2 ±0.11 8.9 ± 0.13 7.5 ± 0.14 8.2 ± 0.007 PQRFa 7.4 ± 0.45 7.6 ± 0.05 7.6 ±0.05 7.6 ± 0.004 BIBP 3226 6.9 ± 0.04 5.9 ± 0.04 7.6 ± 0.04 5.8 ± 0.02

TABLE 4 Activation of intracellular calcium mobilization by COS-7 cellsexpressing human NPFF receptors and Gq/Gz chimera. NPFF1 NPFF2 % of % ofNPPF NPPF Compound pEC50 Response pEC50 Response NPFF 7.8 ± 0.10 100 8.7± 0.02 100 fPP <5.0 0 6.7 ± 0.07 78 PQRF 6.7 ± 0.02 93 7.0 ± 0.04 94amideLocalization

Detection of mRNA coding for rat NPFF1 receptors: mRNA was isolated frommultiple tissues (Table 3) and assayed as described. The distribution ofmRNA encoding rat NPFF1 receptors is widespread throughout the centralnervous system, and structures associated with the nervous system (Table3, FIGS. 14, 15). The highest levels of rNPFF1 mRNA are found in thehypothalamus and the pituitary gland. The protected segment seen withmRNA isolated from the pituitary, adrenal gland and ovary isconsiderably shorter than that seen in other tissue (FIG. 15) andindicates the possibility of splice variants of this receptor.Peripheral organs contain little or no mRNA encoding rNPFF1 with theexception of the testes, ovary, the adrenal medulla and the adrenalcortex. There is good correlation between the distribution determined byRT-PCR and RPA (Table 3, FIGS. 14, 15). RT-PCR detected rat NPFF1 inmore areas than RPA as it is a more sensitive technique.

High levels of mRNA encoding NPFF receptors in the hypothalamus andpituitary, with relatively low expression in most of the other regionsassayed implicates this receptor in neuroendocrine control, as well asthe control of feeding and metabolic regulation. Its presence in otherareas, including the spinal cord, medulla and dorsal root gangliaimplicate NPFF receptors as a potential modulator of pain and/or sensorytransmission. Low levels in the hippocampal formation indicate apossible role in learning and memory.

TABLE 5 Summary of distribution of mRNA coding for rat NPFF1 receptorsRibonuclease protection Potential Tissue RT-PCR assay (RPA) applicationsadrenal + + regulation of cortex steroid hormones adrenal + ++regulation of medulla epinephrine release urinary − − urinary bladderincontinence duodenum +/− − gastrointestinal disorders heart +/− −cardiovascular indications kidney + − electrolyte balance, hypertensionliver +/− − diabetes lung +/− − respiratory disorders, asthma ovary + +reproductive function pancreas +/− NA diabetes, endocrine disordersspleen +/− − immune disorders stomach +/− − gastrointestinal disordersstriated +/− − musculoskeletal muscle disorders testicle +/− +reproductive function uterus +/− − reproductive function vas deferens −− reproductive function whole brain +++ spinal cord ++ ++ analgesia,sensory modulation and transmission amygdala +++ +/− caudate/ ++ −modulation of putamen dopaminergic function cerebellum +++ + motorcoordination cerebral ++ + Sensory and cortex motor integration,cognition DRG + + sensory transmission hippocampus +++ +cognition/memory hypothalamus +++ +++ appetite/obesity, neuroendocrineregulation medulla ++ ++ analgesia, motor coordination olfactory ++ NAolfaction bulb pituitary +++ +++ Endocrine/neuro- endocrine regulationsubstantia ++ +++ Modulation of nigra dopaminergic function superior + −modulation of cervical sympathetic ganglion innervation NA = not assayedLocalization of mRNA Coding for hNPFF2 Receptors Using RT-PCR Detectionof mRNA Coding for hNPFF2 Receptors

mRNA was isolated from multiple tissues (Table 4) and assayed asdescribed. The distribution of mRNA encoding hNPFF2 receptors iswidespread throughout all regions assayed. (Table 4, FIG. 16).

TABLE 6 Distribution of mRNA coding for hNPFF2 receptors Region hNPFF2Potential Implications liver ++ Diabetes kidney ++ Hypertension,electrolyte balance Lung ++ Respiratory disorders, asthma heart ++Cardiovascular indications stomach ++ Gastrointestinal disorders smallintestine ++ Gastrointestinal disorders spleen ++ Immune functionpancreas ++ Diabetes, endocrine disorders striated muscle ++Musculoskeletal disorders pituitary ++ Endocrine/neuroendocrineregulation whole brain ++ amygdala ++ Depression, anxiety, mooddisorders hippocampus ++ Cognition/memory spinal cord ++ Analgesia,sensory modulation and transmission cerebellum ++ Motor coordinationthalamus ++ sensory integration substantia nigra ++ Modulation ofdopaminergic function and motor coordination caudate ++ Modulation ofdopaminergic function fetal brain ++ Developmental disorders fetal lung++ Developmental disorders fetal kidney ++ Developmental disorders fetalliver ++ Developmental disorders HEK-293 cells + HeLa cells − Jurkatcells −Localization of mRNA Coding for Human and Rat NPFF.Results

mRNA was isolated from multiple tissues (listed in Table 7) and assayedas described.

Human NPFF1

Quantitative RT-PCR using a fluorgenic probe demonstrated mRNA encodinghuman NPFF1 RNA to be localized in highest abundance in CNS tissue. AllCNS tissues assayed demonstrate moderate levels of NPFF1 RNA. The broaddistribution of NPFF1 mRNA implies a modulatory role in multiple systemswithin the CNS. Highest levels are found in the spinal cord,hippocampus, amygdala, thalamus and hypothalamus. High levels in thespinal cord and thalamus imply an important role in sensory transmissionor modulation (including nociception). The hippocampal formation andamygdala also contain high levels of NPFF1 mRNA. Localization to thesestructures support the hypothesis that NPFF is involved in themodulation of learning and memory as well as having a role in theregulation of fear, mood, and may provide a target for the treatment ofdepression, anxiety, phobias and mood disorders.

NPFF1 mRNA is also expressed in the hypothalamus in moderate amounts.This suggests a role in neuroendocrine regulation, regulation ofcircadian rhythms, regulation of appetite/feeding behavior and otherfunctions that are modulated by the hypothalamus. NPFF1 mRNA is alsoexpressed, although at somewhat lower levels, in the basal ganglia. Thecaudate-putamen, and substantia nigra both express moderate levels ofNPFF1 mRNA. Localization to these regions implies a role in regulationof dopaminergic systems, and may provide a therapeutic target fortreatment of movement disorders such as Parkinsons disease or tardivedyskinesia. The cerebellum also contains substantial amounts of NPFF1mRNA indicating a role in the control of movement.

Fetal brain, although expressing NPFF1 mRNA, does so in much lowerlevels than that found in the adult. There is a five-fold difference inmRNA levels between fetal and adult brain. It is not known at this timeif the developmental regulation is global within the CNS or restrictedto selected regions. The time course of this increase has not beenexamined and would be important in understanding the function of thisreceptor.

In peripheral tissue, all tissues assayed expressed measurable NPFF1mRNA levels. However, levels in peripheral tissue are much lower thanthose found in the CNS. The peripheral tissues expressing the highestlevels of NPFF1 mRNA are spleen, lung and fetal lung. Levels in thesetissues are more than 10 fold lower than that detected in the highestCNS regions. Others tissues assayed contain low levels of NPFF1 mRNA asindicated in Table 7.

In summary, the distribution of human NPFF1 mRNA implies broadregulatory function in the CNS, most notably in sensory transmission,modulation of the limbic system, modulation of feeding/circadianrhythms, and modulation of extrapyramidal motor systems. Its presence,albeit at low levels in peripheral tissues implies of broad regulatoryrole in multiple organ systems.

Human NPFF2

Unlike the distribution of human NPFF1 mRNA, which is expressedprimarily in the CNS, the highest levels of NPFF2 RNA are found in theplacenta. Expression in the placenta is four-fold higher than any othertissue assayed (Table 7). Presence of high levels NPFF2 receptor mRNA inthe placenta indicates a role in gestational regulation and possiblegestational abnormalities. It is not known at this time, whether NPFF2mRNA is present at all stages of development, or which cells in theplacenta express these receptors. Other tissues expressing NPFF2 mRNAinclude the small intestine, pituitary and spleen. RNA levels in theplacenta are 20 fold higher than in these organs.

Within the CNS, highest levels of NPFF2 mRNA expression are found in theamygdala, caudate-putamen, and the hippocampal formation. These regionsalso express high levels of NPFF1 mRNA. As with NPFF1, localization tolimbic structures supports the hypothesis that NPFF is involved in themodulation of learning and memory as well as having a role in theregulation of fear, mood, and may provide a target for the treatment ofdepression, anxiety, phobias and mood disorders. Localization to thecaudate/putamen implies regulation of dopaminergic systems and a role inthe regulation of extrapyramidal motor systems. Other areas assayed arelisted in Table 7.

In summary, human NPFF2 mRNA is broadly distributed in both CNS andperipheral tissue. This implies broad regulatory functions in multipleorgan systems. High levels in the placenta indicate a significant rolein gestational physiology. Within the CNS, its implied function ismodulation of the limbic system and extrapyramidal motor systems. Itspresence, albeit at low levels in multiple tissues implies a broadmodulatory role involving multiple physiological modalities.

Rat NPFF1

As with the human NPFF1 receptor mRNA, highest levels of rat NPFF1 RNAare found in central nervous system structures. Highest levels are foundin the hypothalamus, amygdala, and the substanta nigra. All CNSstructures assayed express rNPFF1 RNA (Table 8).

The high levels of NPFF1 mRNA expressed in the hypothalamus indicate arole in neuroendocrine regulation, regulation of circadian rhythms,regulation of appetite and other functions that are modulated by thehypothalamus. High levels in the amygdala and other limbic (or limbicrelated) structures suggest a role in modulation of mood, fear, phobia,anxiety and may provide a therapeutic target for the treatment ofdepression and other neuropsychiatric disorders.

The presence of lower levels of NPFF1 RNA in other areas such as thehippocampal formation, spinal cord, medulla, caudate-putamen, cerebralcortex, cerebellum and other areas suggests diverse functions assuggested in Table 8.

The tissue showing the highest levels of NPFF1 mRNA outside the CNS isthe testes. Levels in the testes are more than approximately half of thelevels found in the hypothalamus, and containing approximately the samelevels as those found in the amygdala, substantia nigra, and olfactorybulb (see Table 8). This strongly suggests a role in endocrineregulation or reproductive function. Other peripheral tissues showingmoderate amounts of NPFF1 mRNA are listed in Table 8.

Rat NPFF2

As with rat NPFF1, high levels of rat NPFF2 mRNA are found in CNSstructures. Highest levels are found in the spinal cord and medulla.Localization to these structures as well as moderate levels in thedorsal root and trigeminal ganglia, strongly suggest a role in sensorytransmission (or modulation) including nociceptive stimuli. In additionto the above, there are also moderate levels of NPFF2 RNA localized tothe retina. This suggests a role in modulation of visual stimuli orcircadian rhythms.

Other CNS regions expressing high levels of NPFF2 RNA include thehypothalamus, substantia nigra and amygdala. The high levels of NPFF2mRNA expressed in the hypothalamus indicate a role in neuroendocrineregulation, regulation of circadian rhythms, regulation of appetite andother functions that are modulated by the hypothalamus. High levels inthe amygdala suggests a role in modulation of mood, fear, phobia,anxiety and may provide a therapeutic target for the treatment ofdepression and other neuropsychiatric disorders.

The tissue expressing the highest levels of NPFF2 mRNA outside the CNSis the heart. NPFF2 RNA levels in the heart are comparable to thosefound in many CNS structures. The heart expresses similar levels ofNPFF2 RNA as the spinal cord, medulla, hypothalamus, or substantianigra. Another tissue expressing moderate levels of NPFF2 mRNA is theaorta. This distribution strongly implies regulation of cardiovascularfunction, perhaps by innervation from brain stem autonomic centers. Itis not known if the NPFF2 mRNA is localized to myocytes within the heartor if they are localized on the conductance fibers, smooth muscle, orendothelial cells.

Lower levels of NPFF2 mRNA are localized in multiple tissues though thebody as listed in Table 8. The localization of NPFF2 mRNA to everytissue assayed indicated that this receptor may have broad regulatoryroles in multiple systems.

In summary: The distribution of rat NPFF1 implies a role in limbicfunction as described, and the distribution of NPFF2 implies a role insensory transmission or modulation. The broad distribution of both ofthese receptors in the central nervous system as well as in peripheralorgans, implies a broad regulatory role in multiple physiologicalsystems.

TABLE 7 Distribution of mRNA coding for human NPFF receptors usingqRT-PCR (mRNA encoding NPFF is expressed as % of highest expressingtissue: spinal cord for NPFF 1 and placenta for NPFF2 + SEM) PotentialRegion h-NPFF1 h-NPFF2 applications heart  0.21 + 0.03  0.39 + 0.21Cardiovascular indications kidney  0.67 + 0.11  0.83 + 0.13Hypertension, electrolyte balance liver  0.35 + 0.07  0.21 + 0.04Diabetes lung  6.96 + 0.56  0.71 + 0.09 Respiratory disorders, asthmapancreas  0.23 + 0.06  0.53 + 0.09 Diabetes, endocrine disorderspituitary  2.46 + 0.32  4.65 + 0.47 Endocrine/ neuroendocrine regulationplacenta  0.23 + 0.03   100 + 13.20 Gestational abnormalities small 2.74 + 0.10  4.39 + 0.17 Gastrointestinal intestine disorders spleen 8.08 + 0.55  3.81 + 0.28 Immune disorders stomach  0.55 + 0.06  .095 +0.14 Gastrointestinal disorders striated  1.22 + 0.16  0.78 + 0.14Musculoskeletal muscle disorders amygdala 43.52 + 4.35 27.24 + 1.78Depression, phobias, anxiety, mood disorders caudate- 19.04 + 0.75 9.30 + 1.12 Modulation of putamen dopaminergic function cerebellum20.48 + 2.14 trace Motor coordination hippocampus 44.56 + 5.55  7.39 +0.75 Cognition/memory hypothalamus 20.65 + 0.97  1.58 + 0.02appetite/obesity, neuroendocrine regulation spinal cord   100 + 4.97 1.32 + 0.07 Analgesia, sensory modulation and transmission substantia13.36 + 0.81  0.57 + 0.06 Modulation of nigra dopaminergic function.Modulation of motor coordination. thalamus 29.84 + 3.75  2.24 + 0.27Sensory integration disorders whole brain 21.28 + 1.00  7.89 + 1.12fetal brain  4.24 + 0.33  0.69 + 0.08 Developmental disorders fetal lung 6.01 + 0.89  0.37 + 0.08 Developmental disorders fetal kidney  1.89 +0.23  2.86 + 0.31 Developmental disorders fetal liver trace  0.54 + 0.06Developmental disorders

TABLE 8 Summary of distribution of mRNA coding for rat NPFF receptors(mRNA encoding NPFF is expressed as % of highest expressing tissue:hypothalamus for NPFF 1 and placenta for NPFF2 + SEM) Potential TissuerNPFF1 rNPFF2 applications adipose  2.56 + 0.24 11.72 + 3.17 metabolicdisorders adrenal cortex  2.98 + 0.35  4.70 + 0.44 regulation of steroidhormones adrenal medulla 16.84 + 1.23 trace regulation of epinephrinerelease amygdala 57.09 + 10.25 41.65 + 5.31 depression, phobias,anxiety, mood disorders aorta  1.23 + 0.24 23.83 + 3.70 cardiovascularindications celiac plexus  3.60 + 0.14 12.15 + 1.25 modulation ofautonomic function cerebellum 17.33 + 1.69 10.41 + 1.51 motorcoordination cerebral cortex 21.72 + 0.78 10.99 + 2.29 Sensory and motorintegration, cognition choroid plexus 24.82 + 1.10 29.54 + 6.92regulation of cerebrospinal fluid colon trace  8.38 + 2.72gastrointestinal disorders dorsal root  2.77 + 0.46 38.26 + 3.47 sensoryganglia transmission duodenum trace  5.28 + 0.37 gastrointestinaldisorders heart  3.19 + 0.30 82.32 + 7.97 cardiovascular indicationshippocampus 20.27 + 1.63  8.28 + 2.41 cognition/memory hypothalamus  100 + 6.15 84.26 + 11.01 appetite/ obesity, neuroendocrine regulationkidney  1.03 + 0.23 20.44 + 1.36 electrolyte balance, hypertension liver 1.82 + 0.31  3.20 + 0.42 diabetes lung  3.72 + 0.29 15.88 + 4.35respiratory disorders, asthma medulla 22.44 + 2.21 92.01 + 6.49analgesia, motor coordination nucleus 34.75 + 0.78 10.85 + 1.60regulation of accumbens dopaminergic function, drug addiction,neuropsychiatric disorders olfactory bulb 40.96 + 4.01  9.83 + 4.53olfaction ovary 13.74 + 1.85 12.35 + 2.59 reproductive function pancreastrace trace diabetes, endocrine disorders pineal trace  4.12 + 0.95regulation of melatonin release pituitary 23.58 + 1.81 33.90 + 1.94endocrine/neuroen docrine regulation retina 14.15 + 0.97 40.19 + 2.48visual disorders salivary gland trace 32.93 + 7.48 spinal cord 24.00 +1.41   100 + 5.91 analgesia, sensory modulation and transmission spleentrace trace immune disorders stomach trace 13.90 + 0.69 gastrointestinaldisorders striated muscle trace trace musculoskeletal disorders striatum17.33 + 1.69 16.37 + 4.59 modulation of dopaminergic function, motordisorders substantia 48.82 + 5.54 66.83 + 8.45 modulation of nigradopaminergic function, modulation of motor coordination testes 42.61 +4.71  4.31 + 0.68 reproductive function thalamus  3.14 + 0.25 14.92 +1.92 sensory integration disorders thymus trace 11.53 + 2.92 immunedisorders trigeminal 16.09 + 0.14 56.82 + 2.33 sensory gangliatransmission urinary bladder trace 15.79 + 1.39 urinary incontinenceuterus trace trace reproductive disorders vas deferens trace tracereproductive function whole brain 21.49 + 1.88 23.83 + 2.97Localization of NPFF Receptor Subtypes in the Rat CNSTelencephalon

The cerebral cortex and the amygdala displayed [¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF binding just above background for both the NPFF1and NPFF2 receptors.

In the basal ganglia the globus pallidus was devoid of any specificbinding. [¹²⁵I] [D-Tyr¹-(NMe)Phe³]NPFF binding related to the NPFF2receptor was discretely located in the dorsolateral caudate-putamen andwas completely displaced by frog PP. NPFF1 binding sites were evidentabove background in the accumbens nucleus. Within the septum there was arostrocaudal gradient in binding sites related to NPFF1. The greatestdensity of binding was observed in the more caudal laterodorsal and theintermediate lateral septal nuclei, while rostrally a moderate densitywas observed. Additionally, moderate NPFF1 binding was detected in themedial septum. See Table 9.

Diencephalon

In the thalamus the majority of [¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF binding wasrelated to the NPFF2 receptor subtype. NPFF2 receptors were detected inthe paraventricular and paratenial nuclei, as well as in the reticular,laterodorsal, anterior pretectal, and parafascicular thalamic nuclei. Asignificant density of NPFF1 binding sites were detected in theanterodorsal thalamic nucleus with lower expression in theparaventricular, central medial and ventral nuclei. In the epithalamus,NPFF2 receptors were present in the lateral habenula. See Table 9.

In the hypothalamus, a rostrocaudal gradient of [¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF binding to the NPFF2 receptor was observed in thelateral hypothalamus with the highest density of binding rostrally. Themedial mammillary nucleus also contained considerable NPFF2 receptorbinding while moderate binding was seen in the lateral anteriorhypothalamus. A lower expression of NPFF2 binding sites was observed inthe lateroanterior hypothalamus. NPFF1 binding sites were difficult todetermine in the hypothalamus due to high background levels and thepossible underestimation of NPFF1 binding densities (see Discussion),however, NPFF1 receptor binding sites were detectable in the tubercinereum. See Table 9.

The hippocampal formation did not exhibit any specific [¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF binding in Ammon's horn, although, a moderatenumber of NPFF1 binding sites were observed in the ventral dentategyrus. In other related limbic structures, NPFF1 receptor binding siteswere detected in the bed nucleus of the stria terminalis and thepre/parasubiculum appeared to contain both NPFF1 and NPFF2 receptors.See Table 9.

Mesencephalon

[¹²⁵I] [D-Tyr¹-(NMe)Phe³]NPFF binding to NPFF2 receptors was identifiedin the anterior pretectal nucleus and displayed a dorsal to ventralgradient with the highest density dorsally. NPFF2-receptor binding wasalso observed in the medial pretectal nucleus, posterior intralaminarthalamic nucleus, interstitial nucleus of mlf, substantia nigra, compactpart, interpeduncular nucleus, rostral and caudal linear nuclei ofraphe, dorsal and median raphe nuclei, retrorubral filed, B95-hydroxytryptamine cells, medial and lateral parabrachial nuclei, andthe microcellular tegmental nucleus. Moderate[¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF binding to the NPFF2 receptor was visiblein the dorsal and ventral periaqueductal gray and there was a very weaksignal in the ventral periaqueductal gray related to the NPFF1 receptor.The superior colliculus, pontine nuclei, and the caudal linear raphenucleus contained NPFF1 receptor binding sites, while the parabrachialnucleus exhibited NPFF1 binding sites just above background. See Table9.

Rhombencephalon (Pons/Medulla)

NPFF2 receptor binding sites were evident in the medial vestibular,spinal trigeminal, gigantocelular reticular, Barrington's and ventralcochlear nuclei, in addition to the nucleus of the solitary tract. Thehighest density of NPFF2 binding sites in the rhombencephalon was seenin the region of the facial nerve in the vicinity of the A5noradrenaline cells. Throughout the pons and medulla there was a lowhomogeneous ligand binding just above background which appeared to berelated to the NPFF2 receptor. NPFF1 binding sites were detectable inBarrington's nucleus, the nucleus of the solitary tract, principaltrigeminal nucleus and throughout the reticular formation. See Table 9.

Spinal cord

The dorsal horn displayed the greatest number of [¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF binding sites in the spinal cord. Ligand bindingin the substantia gelatinosa and lamina X was attributed to the NPFF2receptor. NPFF1 binding sites were evident in the spinal cord ventralhorn. See Table 9.

TABLE 9 Distribution of NPFF1 and NPFF2 receptors in the rat CNS RegionrNPFF1 rNPFF2 Potential Application Telencephalon cerebral + +Cognition, sensory cortex and motor integration amygdala + + Emotion andmemory, social behaviors, modulation of autonomic and neuroendocrinesystems vertical + − Memory, modulation of diagonal cholinergic bandtransmission horizontal + − Memory, modulation of diagonal bandcholinergic transmission globus − − pallidus caudate- − + Sensory/motorputamen integration accumbens n. + − Modulation of dopaminergic functionlateral + + Modulation of higher septal n., cognitive functions, dorsalemotions, and autonomic regulation medial septal + − Cognitiveenhancement n. via cholinergic system Diencephalon para- + + ventricularthal. n. central + + medial thalamic n. paratenial − + Modulation ofthalamic n. information to the medial prefrontal cortex anterodorsal + +Modulation of motor thalamic n. information to the cerebral cortex/Eyemovement reticular − + Alertness/sedation thalamic n. laterodorsal − +Emotional expression thalamic n. para- − + Motor and behavioralfascicular responses to pain thal. n. latero- − + anterior hypothal.lateral − + Ingestive behavior, hypothalamus modulation of paintuber + + cinereum supra- − + Circadian rhythm chiasmatic n. medial − +Integration of mammillary n. autonomic or limbic-related functions withmovement lateral − + habenular n. Hippocampal formation Ammon's horn − −ventral + − Cognition/Memory dentate gyrus bed n. stria + + Centralautonomic terminalis system pre/para- + + Modulation of memory subiculumaquisition Mesencephalon anterior − + Mediate visual pretectal n.reflexes/nociception medial − + pretectal n. post. intra- − + laminar n.interstitial − + n. of mlf superior + − Modulation of visual colliculusinformation/spatial localization peri- + + Analgesia aqueductal graysubstantia − + Modulation of DA nigra, function/Motor compact partcoordination substantia − − nigra, reticular part inter- + + Analgesiapeduncular n. rostral − + linear n. raphe caudal linear + + n. raphe redn. − − microcellular − + tegmental n. dorsal raphe − + Analgesia n.median raphe − + n. locus − − coeruleus Barrington's + + Pontinemicturition n. center-urinary bladder function A5 − + Control ofautonomic noradrenergic functions, modulating cell group the perceptionof pain; blood pressure Rhomb- encephalon (Pons/ Medulla) medial − +Maintenance of vestibular n. balance and equilibrium, Modulatingauditory information n. of + + Modulation of solitary gustatory andtract somatosensory information parabrachial + + Modulation of n.visceral sensory information spinal − + Nociception trigeminal n.cerebellum − − giganto- + + Nociception/Analgesia cellular reticular n.ventral − + Modulation of cochlear n. auditory information Spinal corddorsal horn − + Nociception/Analgesia ventral horn + − lamina X − +Nociception, sensory-visceral reflex arcChromosomal Localization

The human NPFF1 gene maps to SHGC-30283 which is localized to chromosome10q21. The human NPFF2 receptor maps to SHGC-24728, which is localizedto chromosome 4q13.2-q13.3. There are minor positional discrepanciesreported in localization between the G3 and the GH4 radiation hybridpanels.

Discussion

The anatomical distribution of the NPFF1 and NPFF2 receptors in the ratCNS was determined by receptor autoradiography using [¹²⁵I][D-Tyr¹-(NMe)Phe³]NPFF at 0.05 nM and making use of subtype selectivedisplacers to directly visualize the individual receptors, NPFF1 andNPFF2. The radioligand exhibits a somewhat higher affinity for the ratNPFF2 subtype (K_(d)=0.22 nM) relative to the rat NPFF1 subtype(K_(d)=0.65 nM). Thus the data may reflect an approximately threefoldunderestimate of the NPFF1 receptor density relative to that for theNPFF2 subtype. [¹²⁵I] [D-Tyr¹-(NMe)Phe³]NPFF binding to the NPFF1receptor was defined as the frog PP-insensitive binding, as thiscompound is highly selective for NPFF2 [pK_(i)=7.3±0.02 at rat NPFF2 and5.4±0.010 at rat NPFF1 (Table 3). Binding to the NPFF2 receptor wasdefined as the BIBP 3226-insensitive binding, as BIBP 3226 is highlyselective for the NPFF1 receptor [pK_(i)=7.6±0.04 at rat NPFF1 and5.8±0.02 at rat NPFF2](Table 3). The results suggest that while bothNPFF1 and NPFF2 receptors are present in the rat CNS, the NPFF2 receptorappears to be the predominantly expressed receptor. NPFF1 and NPFF2receptors are discretely localized in a number of brain nuclei.

NPFF1 receptors were observed to be in cholinergic forebrain regions,namely the nucleus of the diagonal band, the medial and lateral septalnuclei, and the ventral dentate gyrus. NPFF1 binding sites were alsodetected in the superior colliculus and the spinal cord ventral horn.NPFF2 receptors were found to be present in numerous nuclei in thediencephalon, namely the reticular and laterodorsal thalamic nuclei, thesuprachiasmatic, lateroanterior, lateral, and medial mammillaryhypothalmic nuclei. Caudally, NPFF2 receptors were found in the compactpart of the substantia nigra, periaqueductal gray and various raphenuclei. In all levels of the spinal cord, the dorsal horn and lamina Xcontained NPFF2 receptor binding sites.

NPFF-like immunoreactivity (NPFF-LI) has been described in the rat brain(74, 21). The distribution of NPFF-LI in the rat CNS is very limited,the highest levels of immunoreactivity were observed in the hypothalamusand the spinal cord. NPFF-LI neurons were identified in the medialhypothalamus and nucleus of the solitary tract, while immunoreactivefibers were evident in the lateral septal nucleus, amygdala, the lateralhypothalamus, median eminence, bed nucleus of the stria terminalis,nucleus of the diagonal band, nucleus of the solitary tract, the ventralmedulla and the trigeminal complex. NPFF-LI cells and terminals, as wellas the mRNA for both NPFF1 and NPFF2 (Table 8), have been reported to bepresent in the substantia gelatinosa and lamina X at all levels of thespinal cord of rats (75, 21). The distribution of the NPFF1 and NPFF2receptor binding sites correlates well with the distribution of theNPFF-LI neurons and terminals. Additionally, the distribution of theNPFF1 and NPFF2 receptors is concordant with previous reports of theanatomical distribution of NPFF binding sites obtained using [¹²⁵][D-Tyr¹-(NMe)Phe³]NPFF (35) and [¹²⁵I]Y8Fa (76).

Potential Application

NPFF-like peptides have been associated with pain mechanisms, opioidtolerance, autonomic functions, memory and neuroendocrine regulation.

The anatomical distribution of NPFF-LI, NPFF2 mRNA and NPFF2 receptorbinding sites supports the idea of a role for the NPFF2 receptor in theregulation of pain and analgesia, perhaps by modulating the effects ofthe endogenous opioid peptides. NPFF has been shown to attenuate theanalgesic effects of morphine after intrathecal and intraventricularinjection (77) and the localization suggests that this effect bemediated by the NPFF2 receptor. NPFF-LI in the spinal cord is thought tobe mostly of intrinsic origin and NPFF-LI cells in rostral regions ofthe brain do not send descending fibers to the spinal cord (75).Additionally, no NPFF-LI is found in the dorsal root ganglia, and dorsalrhizotomy does not affect NPFF-LI in the dorsal spinal cord (27, 21).NPFF2 mRNA has been identified in DRGs, and this localization mightimply that NPFF2 receptors are located on the primary afferentterminals, possibly mediating neurotransmitter release. NPFF-LI isconcentrated in lamina I/II, the projection site for primary afferentterminals, a region that contains the highest density of NPFF2 bindingsites. In the substantia gelatinosa primary afferents also make contactwith large NPFF-LI nociceptive neurons which in turn project rostrallyto the mesencephalon and thalamus, possibly playing a role in theautonomic and affective responses to pain. NPFF2 mRNA has beenidentified in the spinal cord supporting a role for this receptor in theascending pain pathway. In lamina X there are NPFF-LI fibers possiblyrelated to descending projections from cells originating around thecentral canal. Thus, the NPFF2 receptor may also be involved insensory-visceral reflex arcs.

To further strengthen the concept that the NPFF2 receptor may beinvolved in nociceptive processing, NPFF2 binding sites were localizedin a variety of brain regions known to be involved in nociception andpain modulation, namely the spinal trigeminal nucleus, parabrachialnucleus, gigantocellular reticular nucleus, A5 noradrenergic cell group,dorsal raphe nucleus, periaqueductal gray, lateral hypothalamus, and theparafascicular thalamic nucleus. Injection of an anterograde trace(PHA-L) into the intermediomedial hypothalamus, a site of NPFF-ir cellbodies, supports the concordance between the NPFF2 binding sitedistribution and NPFF-ir terminals in many of these regions (80).Furthermore, NPFF2 mRNA has been identified in the hypothalamus andmedulla. NPFF2 mRNA was also detected in the trigeminal ganglion whichis most likely one of the sources of NPFF2 receptors found in the spinaltrigeminal nucleus (Table 8).

There is some discordance between the localization of NPFF1 mRNA andNPFF1 binding sites in the spinal cord. While there is noautoradiographic evidence for NPFF1 binding sites in the dorsal horn,NPFF1 mRNA has been identified in the spinal cord and DRG (Table 8).This discrepancy might be explained by the expression of the NPFF1receptor on the peripheral terminal projections of the DRG cells or onthe projections of spinal cord neurons to the ventral horn or morerostrally in the brainstem and thalamus. The localization of NPFF1 mRNAin both spinal cord and DRG and with NPFF1 receptor binding sites in theventral horn is consistent with a potential role for the NPFF1 receptorin the processing of nociceptive information.

NPFF-LI fibers are present in several limbic system-related structures,namely the hippocampus, lateral septal nucleus, accumbens nucleus,nucleus of the diagonal band, and bed nucleus of the stria terminalis.The NPFF1 receptor is expressed in these regions. Furthermore, NPFF1mRNA has been detected in the accumbens nucleus, amygdala, andhippocampal formation. On the basis of this localization, a role for theNPFF1 receptor may be to regulate learning and memory and the emotionalstates of fear and anxiety (78). Kavaliers and Colwell (79) have shownthat mice receiving icv injections of IgG from NPFF antiserum acquirespatial tasks more slowly and perform more poorly, while icv NPFFresulted in better acquision of memory. The effect may be associatedwith the hypothalamo-limbic connections containing NPFF (80).

A role for NPFF receptors in regulating sensory information might beindicated by their presence in the relay nuclei of several sensorypathways. It appears that both of the receptors may participate in themodulation of the visual system. NPFF1 receptor binding sites wereobserved in the superior colliculus while NPFF2 receptor binding siteswere detected in the suprachiasmatic nucleus. Both of these regionsreceive afferents from the retina that contains mRNA for NPFF1 andNPFF2. The possibility that the NPFF2 receptor might play a modulatoryrole in circadian rhythms is supported by the localization of NPFF2binding sites in the suprachiasmatic nucleus. The suprachiasmaticnucleus receives direct input from the retina and is thought to beresponsible for the maintenance of circadian rhythms. In the auditorysystem the NPFF2 receptor appears to be a possible modulator. The NPFF2receptor is present in the cochlear and medial vestibular nuclei.

The identification of NPFF receptor binding sites and mRNA for NPFF1 andNPFF2 in various components of the basal ganglia, namely, the accumbensnucleus, the substantia nigra, compact part, and the caudate-putamen,suggests that NPFF receptors may be involved in regulation of thedopaminergic system, although they are not found on dopaminergic neurons(81). Ibotenic acid lesion studies have shown that NPFF receptors in thesubstantia nigra, compact part are on afferent fibers, and thus mayindirectly influence the mesocorticolimbic system. NPFF2 receptors wereidentified in the dorsolateral caudate-putamen, an region whichrepresents the target area for the somatosensory cortex and may beinvolved in sensorimotor integration.

Some of the highest NPFF-LI in the brain was observed in thehypothalamus, one of the main loci for NPFF-LI cell bodies (74). Thelateral hypothalamus is involved in catecholaminergic and serotonergicfeeding systems and plays a role in circadian feeding and spontaneousactivity. The localization of NPFF2 receptor binding sites and mRNA inthis region suggests that the NPFF2 receptor may be involved in theregulation of ingestive behavior. Some of the NPFF2 receptor bindingsites in the hypothalamus may be located presynaptically on projectionsfrom the amygdala since NPFF2 mRNA has been detected in the amygdala(Table 8). In addition, NPFF1 mRNA was detected in the amygdala andhypothalamus (Table 8), suggesting that NPFF1 receptors may also beinvolved in the regulation of ingestive behaviors. While NPFF1 bindingsites were not evident in the hypothalamus, there was a low density ofNPFF1 binding sites seen in the amygdala. The lateral parabrachial andnucleus of the solitary tract are two other brain regions involved inthe regulation of feeding that contain NPFF1 and NPFF2 receptor bindingsites. The origin of dense immunoreactive terminals in these regions isthought to be from the hypothalamus where NPFF1 and NPFF2 mRNA have beenfound, further supporting a potential role for both receptors iningestive behaviors.

Effects of NPFF on Blood Pressure in Normotensive Rats

Results

NPFF (1.0 mg/kg) produced a transient increase in MAP. A similarincrease in blood pressure was evoked by fPP (0.1 mg/kg).

To test whether the response to fPP was mediated by an action at NPFFreceptors or NPY-Y₁ receptors, we first determined a dose of BIBP 3226(NPY-Y₁ antagonist which has relatively high affinity for the rat NPFF1receptor; see Table 9) which was just sufficient to block the pressorresponse to pLeu,Pro-NPY. We then tested this same dose of BIBP 3226against fPP and NPFF. When administered 1 minute before the agonists,the NPY-Y₁ receptor antagonist BIBP 3226 (0.3 mg/kg) completely blockedthe pressor responses to pLeu,Pro-NPY and fPP, and reduced the responseto NPFF by ca 50%.

Discussion

The pharmacological differentiation of NPFF₁ receptor versus the NPFF2receptor may be accomplished by evaluating the effects of NPFF(non-selective NPFF1/NPFF2 receptor agonist) and fPP (agonist at NPFF-2;inactive at NPFF1). When using fPP as such a tool, however, it must beborne in mind that it also activates NPY receptors. In theseexperiments, the pressor effect of fPP was blocked completely by theNPY-Y₁ receptor antagonist BIBP 3226, at a dose of BIBP 3226 which wasjust sufficient to completely block the response to pLeu,Pro-NPY. Sincethe only receptor which is both (1) activated by fPP and (2) blocked byBIBP 3226 is the NPY-Y₁ receptor, we conclude that fPP elevates bloodpressure via activation of NPY-Y₁ receptors, not NPFF receptors. Inaddition, the pressor response to NPFF was also diminished by ca 50% byBIBP 3226, reflecting the higher affinity of BIBP 3226 for the NPFF1receptor relative to the NPFF2 subtype.

Thus, the receptor subtype which mediates the pressor response tointravenously administered NPFF exhibits the following characteristics:(1) insensitive to activation by fPP, and (2) sensitive to blockade byBIBP 3226. This indicates that the pressor response to intravenouslyadministered NPFF is predominately via activation of the NPFF1 subtype.

Effects of NPFF on the Micturition Reflex in Anesthetized Rats.

Results

We found, unexpectedly, that distension-induced rhythmic contractions ofthe rat bladder were inhibited by NPFF (FIG. 21). The disappearance timewas dose-dependently increased between 0.3 to 3.0 mg/kg, i.v. (FIG. 22).Contractions were also inhibited by fPP (0.1 mg/kg; FIG. 23). To testwhether the response to fPP was via an action at NPFF receptors orNPY-Y₁ receptors, fPP was re-tested in the presence of BIBP 3226 (0.3mg/kg, i.v.). In the presence of a concentration of BIBP 3226 sufficientto block fPP's pressor effect (see above), the inhibitory effect of fPPon micturition was not blocked. Furthermore, BIBP 3226 did not block theinhibitory response to NPFF.

Discussion

These results represent the first description of the inhibitory effectof NPFF on the micturition reflex. The effect was dose-dependent,consistent with an interaction between agonist and receptor. Micturitionwas also inhibited by fPP, which has been shown to be an agonist at theNPFF2 receptor, but devoid of agonist activity at the NPFF1 subtype (seeTable 9). The failure of BIBP 3226 to block the effect of fPP onmicturition, in contrast to its actions on blood pressure (see above),indicates that fPP inhibits micturition via activation of NPFF receptorsand not NPY-Y₁ receptors. Therefore, the receptor which mediatesinhibition of the of the micturition reflex by intravenouslyadministered NPFF is one which is activated by both NPFF and fPP,indicating that it is the NPFF2 subtype.

Effects of NPFF on Food Intake in Rats

The intracerebroventricular (ICV) administration of NPFF to rats wasfound to decrease food intake, suggesting a role for NPFF receptors inthe regulation of feeding behavior (Murase et al, 1996). Interestingly,BIBP3226, a selective NPY Y1 receptor antagonist (Doods, et al, 1996),blocks NPY induced feeding via a mechanism that is not related to NPY Y1receptors (Morgan et al, 1998). This notion was based on the observationthat the S-enantiomer of BIBP3226, BIBP3435 is also able to block NPYinduced feeding in spite of the fact that it binds with very lowaffinity (>10,000 nM) to the human and rat NPY Y1 receptors. Similarfindings were reported in a recent study in mice, after NPY-inducedfeeding was inhibited by BIBP3226, but not by another NPY Y1 antagonistGR231118 (Iyengar et al. 1999). This evidence supports the conclusionthat the inhibition of NPY induced feeding by both BIBP3226 and BIBP3435could be better accounted for by the interaction of these two compoundsat a binding site other than the NPY Y1 receptor. As shown in Table 10,BIBP3226, and BIBP3435, bind with high affinity to the cloned NPFF1receptors, and with lower affinity to NPFF2 receptors. Moreover, asshown in Table 11, both BIBP3226, and BIBP3435 are agonists at thecloned rat NPFF2 receptor, with no agonist activity at the cloned ratNPFF1 receptor. Interestingly, both BIBP3226 and BIBP3435 havefunctional potencies at the rat NPFF2 (EC₅₀) that differ only by10-fold, in agreement with the difference in potency of these twocompounds to inhibit NPY-induced feeding (Morgan et al, 1998). Given thefact that the administration of the endogenous agonist NPFF inhibitsfood intake in rats, it is not unexpected that the two synthetic NPFF2receptor agonists BIBP3226 and BIBP3435 also inhibit food intake.

The selective agonist activity of BIBP3226 and BIBP3435 at the clonedrat NPFF2 receptor together with the preferential expression of the mRNAof NPFF2 in rat hypothalamus (see Table 9), a key structure of thecentral nervous system involved in the regulation of feeding behavior,strongly suggest that the actions of NPFF agonists on feeding behaviorare mediated by the NPFF2 subtype. Altogether these observations predictthat NPFF2 receptor agonists could be used as anorectic agents for thetreatment of obesity and eating disorders.

TABLE 10 pKi of NPFF and other synthetic peptides at cloned human andrat NPFF receptors in 293 human embryonic kidney cells (HEK-293 cells)human rat NPFF1 NPFF2 NPFF1 NPFF2 Compound pKi ″ SEM NPFF 8.5 ″ 0.05 8.6″ 0.03 8.5 ″ 0.03 8.2 ″ 0.02 BIBP3226 6.9 ″ 0.04 5.9 ″ 0.04 7.6 ″ 0.045.8 ″ 0.02 BIBP3435 6.9 ″ 0.03 6.3 ″ 0.05 6.9 ″ 0.03 6.2 ″ 0.07

TABLE 11 Functional activity of NPFF and other synthetic peptides atcloned rat NPFF receptors in COS-7 cells rat NPFF2 rat NPFF1 MaximumEC50 Maximum Effect EC50 Effect Compound (nM) (% NPFF) (nM) (% NPFF)NPFF 9.3 100 2.3 100 BIBP3226 >10,000 0 944 67 BIBP3435 >10,000 0 541571

The cloning of the gene encoding NPFF receptors has provided the meansto explore their physiological roles by pharmacologicalcharacterization, and by Northern and in situ mapping of its mRNAdistribution. Further, the availability of the DNA encoding the NPFFreceptors will facilitate the development of antibodies and antisensetechnologies useful in defining the functions of the gene products invivo. Antisense oligonucleotides which target mRNA molecules toselectively block translation of the gene products in vivo have beenused successfully to relate the expression of a single gene with itsfunctional sequelae. Thus, the cloning of these receptor genes providesthe means to explore their physiological roles in the nervous system andelsewhere, and may thereby help to elucidate structure/functionrelationships within the GPCR superfamily.

REFERENCES

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1. A process involving competitive binding for identifying a chemicalcompound which specifically binds to a human NPFF1 receptor whichcomprises separately contacting cells expressing on their cell surfacethe human NPFF1 receptor, or a membrane preparation of such cells,wherein such cells prior to being transfected with DNA encoding thehuman NPFF1 receptor do not express the human NPFF1 receptor, with boththe chemical compound and a second chemical compound known to bind tothe receptor, and with only the second chemical compound, underconditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the human NPFF1 receptor, adecrease in the binding of the second chemical compound to the humanNPFF1 receptor in the presence of the chemical compound indicating thatthe chemical compound binds to the human NPFF1 receptor, wherein thehuman NPFF1 receptor has an amino acid sequence identical to the aminoacid sequence shown in 1) SEQ ID NO: 8 or 2) that encoded by plasmidpcDNA3.1-hNPFF1 (ATCC Accession No. 203605).
 2. The process of claim 1,wherein the cell is an insect cell.
 3. The process of claim 1, whereinthe cell is a mammalian cell.
 4. The process of claim 3, wherein thecell is non-neuronal in origin.
 5. The process of claim 4, wherein thenon-neuronal cell is a COS-7 cell, a 294 human embryonic kidney cell, aCHO cell, an NIH-3T3 cell, a mouse Y1 cell, or an LM(tk−) cell.
 6. Theprocess of claim 5, wherein the compound is not previously known to bindto a human NPFF1 receptor.
 7. A method of screening a plurality ofchemical compounds not known to bind to a human NPFF1 receptor toidentify a compound which specifically binds to the human NPFF1receptor, which comprises (a) contacting cells transfected with andexpressing DNA encoding the human NPFF1 receptor, or a membranepreparation of such cells, wherein such cells prior to being transfectedwith such DNA do not express the human NPFF1 receptor, with a compoundknown to bind specifically to the human NPFF1 receptor; (b) contactingthe cells of step (a), or a membrane preparation of the cells of step(a), with the plurality of compounds not known to bind specifically tothe human NPFF1 receptor, under conditions permitting binding ofcompounds known to bind to the human NPFF1 receptor; (c) determiningwhether the binding of the compound known to bind to the human NPFF1receptor is reduced in the presence of any compound within the pluralityof compounds, relative to the binding of the compound in the absence ofthe plurality of compounds; and if so (d) separately determining thebinding to the human NPFF1 receptor of compounds included in theplurality of compounds, so as to thereby identify the compound whichspecifically binds to the human NPFF1 receptor; wherein the human NPFF1receptor has an amino acid sequence identical to the amino acid sequenceshown in 1) SEQ ID NO: 8 or 2) that encoded by plasmid pcDNA3.1-hNPFF1(ATCC Accession No. 203605).
 8. A process for determining whether achemical compound specifically binds to and inhibits activation of ahuman NPFF1 receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the human NPFF1 receptor, wherein such cells prior to beingtransfected with DNA encoding the human NPFF1 receptor do not expressthe human NPFF1 receptor, with both the chemical compound and a secondchemical compound known to activate the human NPFF1 receptor, and withonly the second chemical compound, under conditions suitable foractivation of the human NPFF1 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change in the second messenger response inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compoundindicating that the chemical compound inhibits activation of the humanNPFF1 receptor, wherein the human NPFF1 receptor has an amino acidsequence identical to the amino acid sequence shown in 1) SEQ ID NO: 8or 2) that encoded plasmid pcDNA3.1-hNPFF1 (ATCC Accession No. 203605).9. The process of claim 8, wherein the second messenger responsecomprises chloride channel activation and the change in second messengerresponse is a smaller increase in the level of inward chloride currentin the presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compound. 10.The process of claim 8, wherein the cell is an insect cell.
 11. Theprocess of claim 8, wherein the cell is a mammalian cell.
 12. Theprocess of claim 11, wherein the mammalian cell is normeuronal inorigin.
 13. The process of claim 12, wherein the non-neuronal cell is aCOS-7 cell, a CHO cell, a 293 human embryonic kidney cell, an NIH-3T3cell or an LM(tk−) cell.
 14. The process of claim 8, wherein thecompound is not previously known to bind to a human NPFF1 receptor. 15.A method of screening a plurality of chemical compounds not known toinhibit the activation of a human NPFF1 receptor to identify a compoundwhich inhibits the activation of the human NPFF1 receptor, whichcomprises: (a) contacting cells transfected with and expressing thehuman NPFF1 receptor with the plurality of compounds in the presence ofa known human NPFF1 receptor agonist, under conditions permittingactivation of the human NPFF1 receptor; (b) determining whether theactivation of the human NPFF1 receptor is reduced in the presence of theplurality of compounds, relative to the activation of the human NPFF1receptor in the absence of the plurality of compounds; and if so (c)separately determining the inhibition of activation of the human NPFF1receptor for each compound included in the plurality of compounds, so asto thereby identify the compound which inhibits the activation of thehuman NPFF1 receptor; wherein the human NPFF1 receptor has an amino acidsequence identical to the amino acid sequence shown in 1) SEQ ID NO: 8or 2) that encoded by plasmid pcDNA3.1-hNPFF1 (ATCC Accession No.203605).
 16. The process of claim 15, wherein the cell is a mammaliancell.
 17. The process of claim 16, wherein the mammalian cell isnon-neuronal in origin.
 18. The process of claim 17, wherein thenon-neuronal cell is a COS-7 cell, a CHO cell, a 293 human embryonickidney cell, an NIH-3T3 cell or an LM(tk−) cell.